Compositions and Methods for Treatment of Cancer

ABSTRACT

The present invention provides compositions and methods for treating cancer in a human. The invention includes relates to administering a genetically modified T cell to express a CAR wherein the CAR comprises an antigen binding domain, a transmembrane domain, a costimulatory signaling region, and a CD3 zeta signaling domain.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.61/421,470, filed Dec. 9, 2010, and U.S. Provisional Application No.61/502,649, filed Jun. 29, 2011, all of which are hereby incorporatedherein by reference in their entireties.

BACKGROUND OF THE INVENTION

The large majority of patients having B-cell malignancies, includingchronic lymphocytic leukemia (CLL), will die from their disease. Oneapproach to treating these patients is to genetically modify T cells totarget antigens expressed on tumor cells through the expression ofchimeric antigen receptors (CARs). CARs are antigen receptors that aredesigned to recognize cell surface antigens in a human leukocyteantigen-independent manner. Attempts in using genetically modified cellsexpressing CARs to treat these types of patients have met with verylimited success. See for example, Brentjens et al., 2010, MolecularTherapy, 18:4, 666-668; Morgan et al., 2010, Molecular Therapy,published online Feb. 23, 2010, pages 1-9; and, Till et al., 2008,Blood, 112:2261-2271.

In most cancers, tumor-specific antigens are not yet well defined, butin B cell malignancies, CD19 is an attractive tumor target. Expressionof CD19 is restricted to normal and malignant B cells (Uckun, et al.Blood, 1988, 71:13-29), so that CD19 is a widely accepted target tosafely test CARs. While CARs can trigger T-cell activation in a mannersimilar to an endogenous T-cell receptor, a major impediment to theclinical application of this technology to date has been limited in vivoexpansion of CAR+ T cells, rapid disappearance of the cells afterinfusion, and disappointing clinical activity (Jena, et al., Blood,2010, 116:1035-1044; Uckun, et al. Blood, 1988, 71:13-29).

Thus, there is an urgent need in the art for compositions and methodsfor treatment of cancer using CARs that can expand in vivo. The presentinvention addresses this need.

SUMMARY OF THE INVENTION

The present invention provides an isolated nucleic acid sequenceencoding a chimeric antigen receptor (CAR), wherein the CAR comprises anantigen binding domain, a transmembrane domain, a costimulatorysignaling region, and a CD3 zeta signaling domain, wherein the CD3 zetasignaling domain comprises the amino acid sequence of SEQ ID NO: 24.

In one embodiment, the nucleic acid sequence encodes a CAR comprisingthe amino acid sequence of SEQ ID NO: 12.

In one embodiment, the nucleic acid sequence encoding a CAR comprisesthe nucleic acid sequence of SEQ ID NO: 8.

In one embodiment, the antigen binding domain in the CAR is an antibodyor an antigen-binding fragment thereof. Preferably, the antigen-bindingfragment is a Fab or a scFv.

In one embodiment, the antigen binding domain in the CAR binds to atumor antigen. In one embodiment, the tumor antigen is associated with ahematologic malignancy. In another embodiment, the tumor antigen isassociated with a solid tumor. In yet another embodiment, the tumorantigen is selected from the group consisting of CD19, CD20, CD22, ROR1,mesothelin, CD33/IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRvIII, GD-2,NY-ESO-1 TCR, MAGE A3 TCR, and any combination thereof.

In one embodiment, the costimulatory signaling region in the CARcomprises the intracellular domain of a costimulatory molecule selectedfrom the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1,ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, B7-H3, a ligand that specifically binds with CD83, and anycombination thereof.

In one embodiment, the CD3 zeta signaling domain in the CAR is encodedby the nucleic acid sequence of SEQ ID NO: 18.

The invention also provides an isolated CAR comprising an antigenbinding domain, a transmembrane domain, a costimulatory signalingregion, and a CD3 zeta signaling domain, wherein the CD3 zeta signalingdomain comprises the amino acid sequence of SEQ ID NO: 24.

The invention also provides a cell comprising a nucleic acid sequenceencoding a CAR, wherein the CAR comprises an antigen binding domain, atransmembrane domain, a costimulatory signaling region, and a CD3 zetasignaling domain comprising the amino acid sequence of SEQ ID NO: 24.

In one embodiment, the cell comprising the CAR is selected from thegroup consisting of a T cell, a Natural Killer (NK) cell, a cytotoxic Tlymphocyte (CTL), and a regulatory T cell.

In one embodiment, the cell comprising the CAR exhibits an anti-tumorimmunity when the antigen binding domain of the CAR binds to itscorresponding antigen.

The invention also provides a vector comprising a nucleic acid sequenceencoding a CAR, wherein the CAR comprises an antigen binding domain, acostimulatory signaling region, and a CD3 zeta signaling domain, whereinthe CD3 zeta signaling domain comprises the amino acid sequence of SEQID NO: 24.

The invention also provides a method for stimulating a T cell-mediatedimmune response to a target cell population or tissue in a mammal. Inone embodiment, the method comprises administering to a mammal aneffective amount of a cell genetically modified to express a CAR whereinthe CAR comprises an antigen binding domain, a costimulatory signalingregion, and a CD3 zeta signaling domain comprising the amino acidsequence of SEQ ID NO: 24, wherein the antigen binding domain isselected to specifically recognize the target cell population or tissue.

The invention also provides a method of providing an anti-tumor immunityin a mammal. In one embodiment, the method comprises administering to amammal an effective amount of a cell genetically modified to express aCAR wherein the CAR comprises an antigen binding domain, a costimulatorysignaling region, and a CD3 zeta signaling domain comprising the aminoacid sequence of SEQ ID NO: 24, thereby providing an anti-tumor immunityin the mammal.

The invention also includes a method of treating a mammal having adisease, disorder or condition associated with an elevated expression ofa tumor antigen. In one embodiment, the method comprises administeringto a mammal an effective amount of a cell genetically modified toexpress a CAR wherein the CAR comprises an antigen binding domain, acostimulatory signaling region, and a CD3 zeta signaling domaincomprising the amino acid sequence of SEQ ID NO: 24, thereby treatingthe mammal.

In one embodiment, the cell is an autologous T cell.

In one embodiment, the tumor antigen is selected from the groupconsisting of CD19, CD20, CD22, ROR1, mesothelin, CD33/IL3Ra, c-Met,PSMA, Glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, and anycombination thereof.

The invention also provides a method of treating a human with chroniclymphocytic leukemia. In one embodiment, the method comprisesadministering to a human a T cell genetically engineered to express aCAR wherein the CAR comprises an antigen binding domain, a costimulatorysignaling region, and a CD3 zeta signaling domain comprising the aminoacid sequence of SEQ ID NO: 24.

In one embodiment, the human is resistant to at least onechemotherapeutic agent

In one embodiment, the chronic lymphocytic leukemia is refractory CD19+leukemia and lymphoma.

The invention also includes a method of generating a persistingpopulation of genetically engineered T cells in a human diagnosed withcancer. In one embodiment, the method comprises administering to a humana T cell genetically engineered to express a CAR wherein the CARcomprises an antigen binding domain, a costimulatory signaling region,and a CD3 zeta signaling domain comprising the amino acid sequence ofSEQ ID NO: 24, wherein the persisting population of geneticallyengineered T cells persists in the human for at least one month afteradministration.

In one embodiment, the persisting population of genetically engineered Tcells comprises at least one cell selected from the group consisting ofa T cell that was administered to the human, a progeny of a T cell thatwas administered to the human, and a combination thereof.

In one embodiment, the persisting population of genetically engineered Tcells comprises a memory T cell.

In one embodiment, the persisting population of genetically engineered Tcells persists in the human for at least three months afteradministration. In another embodiment, the persisting population ofgenetically engineered T cells persists in the human for at least fourmonths, five months, six months, seven months, eight months, ninemonths, ten months, eleven months, twelve months, two years, or threeyears after administration.

In one embodiment, the chronic lymphocytic leukemia is treated.

The invention also provides a method of expanding a population ofgenetically engineered T cells in a human diagnosed with cancer. In oneembodiment, the method comprises administering to a human a T cellgenetically engineered to express a CAR wherein the CAR comprises anantigen binding domain, a costimulatory signaling region, and a CD3 zetasignaling domain comprising the amino acid sequence of SEQ ID NO: 24,wherein the administered genetically engineered T cell produces apopulation of progeny T cells in the human.

In one embodiment, the progeny T cells in the human comprise a memory Tcell.

In one embodiment, the T cell is an autologous T cell.

In another embodiment, the human is resistant to at least onechemotherapeutic agent.

In one embodiment, the cancer is chronic lymphocytic leukemia. Inanother embodiment, the chronic lymphocytic leukemia is refractory CD19+leukemia and lymphoma.

In one embodiment, the population of progeny T cells persists in thehuman for at least three months after administration. In anotherembodiment, the population of progeny T cells persist in the human forat least four months, five months, six months, seven months, eightmonths, nine months, ten months, eleven months, twelve months, twoyears, or three years after administration.

In one embodiment, the cancer is treated.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1, comprising FIGS. 1A through 1C, is a series of images of theschematic representations of the gene-transfer vector and transgene,gene modified T cell manufacturing and clinical protocol design. FIG. 1Adepicts the lentiviral vectors and transgene that show the majorfunctional elements. A vesicular stomatitis virus protein G pseudotypedclinical grade lentiviral vector (designated pELPs 19BBz) directingexpression of anti-CD19 scFv derived from FMC63 murine monoclonalantibody, human CD8α hinge and transmembrane domain, and human 4-1BB andCD3zeta signaling domains was produced. Constitutive expression of thetransgene was directed by inclusion of an EF-1α (elongation factor-1αpromoter); LTR, long terminal repeat; RRE, rev response element. (cPPT)and the central termination sequence (CTS). Figure is not to scale. FIG.1B depicts T cell manufacturing. Autologous cells were obtained via anapheresis, and T cells were enriched by mononuclear cell elutriation,washed and residual leukemia cells depleted by addition of anti-CD3/CD28coated paramagnetic beads for positive selection and activation of Tcells. Lentiviral vector was added at the time of cell activation andwas washed out on day 3 post culture initiation. Cells were expanded ona rocking platform device (WAVE Bioreactor System) for 8-12 days. On thefinal day of culture the beads were removed by passage over a magneticfield and the CART19 T cells harvested and cryopreserved in infusiblemedium. FIG. 1C depicts the clinical protocol design. Patients weregiven lymphodepleting chemotherapy as described, followed by CART19infusion #1 by i.v. gravity flow drip over a period of 15-20 minutes.The infusion was given using a split dose approach over 3 days (100/%,30%, 60%) beginning 1 to 5 days after completion of chemotherapy.Endpoint assays were conducted on study week 4. At the conclusion ofactive monitoring, subjects were transferred to a destination protocolfor long term follow up as per FDA guidance.

FIG. 2, comprising FIGS. 2A through 2F, is a series of imagesdemonstrating sustained in vivo expansion and persistence in blood andmarrow of CART19 cells. DNA isolated from whole blood as depicted inFIG. 2A through 2C or marrow as depicted in FIG. 2D through 2F, samplesobtained from UPN 01 as depicted in FIGS. 2A and 2D, UPN 02 as depictedin FIGS. 2B and 2E and UPN 03 as depicted in FIGS. 2C and 2F wassubjected in bulk to Q-PCR analysis using a qualified assay to detectand quantify CART19 sequences. Each data point represents the average oftriplicate measurements on 100-200 ng genomic DNA, with maximal % CVless than 1.56%. Pass/fail parameters for the assay includedpre-established ranges for slope and efficiency of amplification, andamplification of a reference sample. The lower limit of quantificationfor the assay established by the standard curve range was 2 copiestransgene/microgram genomic DNA: sample values below that number areconsidered estimates and presented if at least ⅔ replicates generated aCt value with % CV for the values 15%. CART19 cells were infused at day0, 1, and 2 for UPN 01 and UPN 03, and days 0, 1, 2 and 11 for UPN 02.

FIG. 3, comprising FIGS. 3A through 3D, is a series of imagesdemonstrating serum and bone marrow cytokines before and after CAR Tcell infusion; longitudinal measurements of changes in serum cytokines,chemokines and cytokine receptors in UPN 01 as depicted in FIG. 3A, UPN02 as depicted in FIG. 3B and UPN 03 as depicted in FIG. 3C, on theindicated day after CART19 cell infusion and serial assessments of thesame analytes in the bone marrow from UPN 03 as depicted in FIG. 3D.Samples were subjected multiplex analysis using Luminex bead arraytechnology and pre-assembled and validated multiplex kits. Analytes witha >=3 fold change are indicated, and plotted as relative change frombaseline as depicted in FIG. 3A through 3C or as absolute values asdepicted in FIG. 3D. Absolute values for each analyte at each time-pointwere derived from a recombinant protein-based standard curve over a3-fold 8-point dilution series, with upper and lower limits ofquantification (ULOQ, LLOQ) determined by the 80-120% observed/expectedcutoff values for the standard curves. Each sample was evaluated induplicate with average values calculated and % CV in most cases lessthan 10%. To accommodate consolidated data presentation in the contextof the wide range for the absolute values, data are presented asfold-change over the baseline value for each analyte. In cases wherebaseline values were not detectable, half of the lowest standard curvevalue was used as the baseline value. Standard curve ranges for analytesand baseline (day 0) values (listed in parentheses sequentially forUPN01, 02 and 03), all in pg/ml: IL1-Rα: 35.5-29,318 (689, 301, 287);IL-6: 2.7-4,572 (7, 10.1, 8.7); IFN-γ: 11.2-23,972 (2.8, ND, 4.2);CXCL10: 2.1-5,319 (481, 115, 287); MIP-1β: 3.3-7,233 (99.7, 371, 174);MCP-1: 4.8-3,600 (403, 560, 828); CXCL9: 48.2-3,700 (1,412, 126, 177);IL2-Rα: 13.4-34,210 (4,319, 9,477, 610); IL-8: 2.4-5,278 (15.3, 14.5,14.6); IL-10: 6.7-13,874 (8.5, 5.4, 0.7); MIP-1α: 7.1-13,778 (57.6,57.3, 48.1).

FIG. 4, comprising FIGS. 4A through 4D, is a series of images depictingprolonged surface CART19 expression and establishment of functionalmemory CARs in vivo. FIG. 4A depicts detection of CAR-expressing CD3+lymphocytes and absence of B cells in periphery and marrow. Freshlyprocessed peripheral blood or marrow mononuclear cells obtained from UPN03 at day 169 post-CART19 cell infusion were evaluated by flow-cytometryfor surface expression of CAR19 (top) or presence of B cells (bottom);as a control, PBMC obtained from a healthy donor ND365 were stained. Thegating strategy for the CD3+ and B cell populations is presented in FIG.9. To evaluate CAR19 expression in CD3+ lymphocytes, samples wereco-stained with antibodies to CD14-PE-Cy7 and CD16-PE-Cy7 (dump channel)and CD3-FITC, positively gated on CD3+, and evaluated for CAR19expression in the CD8+ and CD8-lymphocyte compartments by co-stainingwith CD8a-PE and the anti-CAR19 idiotype antibody conjugated toAlexa-647. Data in plots are gated on the dumpchannel-negative/CD3-positive cell population. To evaluate the presenceof B cells, samples were co-stained with antibodies to CD14-APC andCD3-FITC (dump channels) and evaluated for the presence of B cells inthe dump channel-negative fraction by co-staining with antibodies toCD20-PE and CD19-PE-Cy-7. In all cases, negative gate quadrants wereestablished on no-stain controls as depicted in FIGS. 4B and 4C. T cellimmunophenotyping of CD4+(FIG. 4B) and CD8+(FIG. 4C) T cell subsets isshown. Frozen peripheral blood samples from UPN 03 obtained by apheresisat day 56 and 169 post T cell infusion were rested overnight in culturemedium with no added factors, washed, and subjected to multi-parametricimmunophenotyping for expression of markers of T cell memory,activation, and exhaustion. The gating strategy, as depicted in FIG. 8,involved an initial gating on dump channel (CD14, CD16, Live/DeadAqua)-negative and CD3-positive cells, followed by positive gates onCD4+ and CD8+ cells. Gates and quadrants were established using FMOcontrols (CAR, CD45RA, PD-1, CD25, CD127, CCR7) or by gating on positivecell populations (CD3, CD4, CD8) and clearly delineated subsets (CD27,CD28, CD57); data were displayed after bi-exponential transformation forobjective visualization of events. FIG. 4D depicts functional competenceof persisting CAR cells. Frozen peripheral blood samples from UPN 03obtained by apheresis at day 56 and 169 post T cell infusion were restedovernight in culture medium with no added factors, washed, and evaluateddirectly ex-vivo for the ability to recognize CD19-expressing targetcells using CD107 degranulation assays. Following a two-hour incubationin the presence of anti-CD28, anti-CD49d, and CD107-FITC, cell mixtureswere harvested, washed, and subjected to multi-parametric flowcytometric analysis to evaluate the ability of CART19 cells tode-granulate in response to CD19-expressing targets. The gating strategyinvolved an initial gate on dump channels (CD14-PE-Cy7, CD16-PE-Cy7,Live/Dead Aqua)-negative and CD3-PE-positive cells, followed by gatingon CD8-PE-Texas Red-positive cells; presented data is for the CD8+ gatedpopulation. In all cases, negative gate quadrants were established onno-stain controls.

FIG. 5, comprising FIGS. 5A through 5C, is series of images depictingthe results of experiments evaluating clinical responses after infusionof CART19 cells. FIG. 5A depicts that UPN 02 was treated with two cyclesof rituximab and bendamustine with minimal response (R/B, arrow). CART19T cells were infused beginning 4 days after bendamustine only (B,arrow). The rituximab and bendamustine-resistant leukemia was rapidlycleared from blood, as indicated by a decrease in the absolutelymphocyte count (ALC) from 60.600/μl to 200/μl within 18 days of theinfusion. Corticosteroid treatment was started on day 18 post infusiondue to malaise and non-infectious febrile syndrome. The reference line(dotted) indicates upper limit of normal for ALC. FIG. 5B depicts theresults of example experiments staining sequential bone marrow biopsy orclot specimens from patient UPN 01 and 03 for CD20. Pretreatmentinfiltration with leukemia present in both patients was absent on posttreatment specimens accompanied by normalization of cellularity andtrilineage hematopoiesis. UPN 01 has not had any CLL cells detected asassessed by flow cytometry, cytogenetics and fluorescence in-situhybridization or normal B cells detected by flow cytometry in bonemarrow or blood. UPN 03 had 5% residual normal CD5-negative B cellsconfirmed by flow cytometry on day +23, which also showed them to bepolyclonal; no normal B cells were detected at day +176. FIG. 5C depictsthe results of experiments using sequential CT imaging to assess therapid resolution of chemotherapy-resistant generalized lymphadenopathy.Bilateral axillary masses resolved by 83 (UPN 01) and 31 (UPN 03) dayspost infusion, as indicated by arrows and circle.

FIG. 6, comprising FIGS. 6A through 6C, is a series of images depictingabsolute lymphocyte counts and total CART19+ cells in circulation forUPN 01, 02, 03. The total number of lymphocytes (Total normal and CLLcells) vs. Total CART19+ cells in circulation is plotted for all 3subjects using the absolute lymphocyte count from CBC values, andassuming a 5.0 L volume of blood. The total number of CART19 cells incirculation was calculated by using the tandem CBC values with absolutelymphocyte counts and the Q-PCR marking values as depicted in FIG. 2,converting copies/μg DNA to average % marking as described elsewhereherein. The Q-PCR % marking was found to correlate closely (<2 foldvariation) with the flow cytometric characterization of the infusionproducts and with data from samples where concomitant flow cytometrydata was available to directly enumerate CART19 cells by staining.

FIG. 7, comprising FIGS. 7A through 7D is a series of images depictingexperiments involving the direct ex vivo detection of CART19-positivecells in UPN-01 PBMC 71 days post-T cell infusion. UPN-01 PBMC collectedeither fresh post-apheresis on day 71 day post infusion, or frozen atthe time of apheresis for manufacture of the T cell product (baseline)and viably thawed prior to the staining, were subjected toflow-cytometric analysis to detect the presence of CART19 cells thatexpress the CAR19 moiety on the surface. To evaluate the expression ofCAR19 in lymphocytes, samples were co-stained with CD3-PE and theanti-CAR19 idiotype antibody conjugated to Alexa-647, or co-stained withCD3-PE alone (FMO for CAR19). FIG. 7A depicts that an initial lymphocytegate was established based on forward and side scatter (FSC vs SSC),followed by gating on CD3+ cells. FIG. 7B depicts CD3+ lymphocyte gate;FIG. 7C depicts CAR idiotype stain; FIG. 7D depicts CAR idiotype FMO.The CAR19-positive gate was established on the CAR19 FMO samples.

FIG. 8, comprising FIGS. 8A through 8C, is a series of images depictingthe gating strategy to identify CART19 expression by using polychromaticflow cytometry in UPN 03 blood specimens. The gating strategy for FIG.8C is shown for the UPN 03 Day 56 sample and is representative of thestrategy used on the UPN 03 Day 169 sample. FIG. 8A depicts primarygate: Dump (CD14, CD16, LIVE/dead Aqua) negative, CD3-positive. FIG. 8Bdepicts secondary gates: CD4-positive, CD8positive. FIG. 8C depictstertiary gates: CAR19-positive and CAR19-negative, established on CARFMO samples (right-most panels).

FIG. 9 depicts the gating strategy to directly identify CART19expression and B cells in blood and marrow specimens. The gatingstrategy for FIG. 4A, which shows detection of CAR-expressing CD3+lymphocytes and absence of B cells in periphery and marrow: Leftplot:Cell gate: Upper panel: positive gate for CD3+ cells, Lower panel:negative gate (CD14-negative, CD3-negative) for B cells. NC365,peripheral blood control cells from a healthy donor

FIG. 10 is an image summarizing the patient demographics and response.

FIG. 11 depicts the manufacturing process of CART-19 cells

FIG. 12, comprising FIGS. 12A through 12D, is a series of imagesdepicting the clinical response in a patient. FIG. 12A shows thelentiviral vector used to infect T cells from the patient. Apseudotyped, clinical-grade lentiviral vector of vesicular stomatitisvirus protein G (pELPs 19-BB-z) directing expression of anti-CD19 scFvderived from FMC63 murine monoclonal antibody, human CD8α hinge andtransmembrane domain, and human 4-1BB and CD3 signaling domains wasproduced. Details of the CAR19 transgene, at the bottom of FIG. 12A,show the major functional elements. The figure is not to scale. 3′LTRdenotes 3′ long terminal repeat; 5′LTR, 5′ long terminal repeat; Amp R,ampicillin resistance gene; Bovine GH Poly A, bovine growth hormone withpolyadenylation tail; cPPT/CTS, central polypurine tract with centraltermination sequence; EF-1α, elongation factor 1-alpha; env, envelope;gag, group-specific antigen; pol, HIV gene encoding polymerase andreverse transcriptase; R, repeat; RRE, rev response element; scFv,single-chain variable fragment; TM, transmembrane; and WPRE, woodchuckhepatitis virus post-transcriptional regulatory element. FIG. 12B showsserum creatinine, uric acid, and lactate dehydrogenase (LDH) levels fromday 1 to day 28 after the first CART19-cell infusion. The peak levelscoincided with hospitalization for the tumor lysis syndrome. FIG. 12Cshows bone marrow-biopsy specimens obtained 3 days after chemotherapy(day −1, before CART19-cell infusion) and 23 days and 6 months afterCART19-cell infusion (hematoxylin and eosin). The baseline specimenshows hypercellular bone marrow (60%) with trilineage hematopoiesis,infiltrated by predominantly interstitial aggregates of small, maturelymphocytes that account for 40% of total cellularity. The specimenobtained on day 23 shows residual lymphoid aggregates (10%) that werenegative for chronic lymphoid leukemia (CLL), with a mixture of T cellsand CD5-negative B cells. The specimen obtained 6 months after infusionshows trilineage hematopoiesis, without lymphoid aggregates andcontinued absence of CLL. FIG. 12D shows contrast-enhanced CT scansobtained before the patient was enrolled in the study and 31 days and104 days after the first infusion. The preinfusion CT scan reveals1-to-3-cm bilateral masses. Regression of axillary lymphadenopathyoccurred within 1 month after infusion and was sustained. Arrowshighlight various enlarged lymph nodes before therapy and lymph-noderesponses on comparable CT scans after therapy.

FIG. 13, comprising FIGS. 13A through 13E, is a series of imagesdepicting serum and bone marrow cytokines before and after chimericantigen receptor T-cell infusion. Serial measurements of the cytokineinterferon-γ (FIG. 13A), the interferon-γ-stimulated chemokines C-X-Cmotif chemokine 10 (CXCL10) (FIG. 13B) and C-X-C motif ligand 9 (CXCL9)(FIG. 13C), and interleukin-6 (FIG. 13D) were measured at the indicatedtime points. The increases in these inflammatory cytokines andchemokines coincided with the onset of the tumor lysis syndrome. Lowlevels of interleukin-6 were detected at baseline, whereas interferon-γ.CXCL9, and CXCL10 were below the limits of detection at baseline.Standard-curve ranges for the analytes and baseline values in thepatient, given in parentheses, were as follows: interferon-γ, 11.2 to23,972 pg per milliliter (1.4 pg per milliliter); CXCL10, 2.1 to 5319 pgper milliliter (274 pg per milliliter); CXCL9, 48.2 to 3700 pg permilliliter (177 pg per milliliter); interleukin-6, 2.7 to 4572 pg permilliliter (8.3 pg per milliliter); tumor necrosis factor α (TNF-α), 1.9to 4005 pg per milliliter (not detectable); and soluble interleukin-2receptor, 13.4 to 34,210 pg per milliliter (644 pg per milliliter). FIG.13E shows the induction of the immune response in bone marrow. Thecytokines TNF-α, interleukin-6, interferon-γ, chemokine CXCL9, andsoluble interleukin-2 receptor were measured in supernatant fluidsobtained from bone marrow aspirates on the indicated days before andafter CART19-cell infusion. The increases in levels of interleukin-6,interferon-γ, CXCL9, and soluble interleukin-2 receptor coincided withthe tumor lysis syndrome, peak chimeric antigen receptor T-cellinfiltration, and eradication of the leukemic infiltrate.

FIG. 14, comprising FIGS. 14A through 14C, is a series of imagesdepicting expansion and persistence of chimeric antigen receptor T cellsin vivo. Genomic DNA (gDNA) was isolated from samples of the patient'swhole blood (FIG. 14A) and bone marrow aspirates (FIG. 14B) collected atserial time points before and after chimeric antigen receptor T-cellinfusion and used for quantitative real-time polymerase-chain-reaction(PCR) analysis. As assessed on the basis of transgenic DNA and thepercentage of lymphocytes expressing CAR19, the chimeric antigenreceptor T cells expanded to levels that were more than 1000 times ashigh as initial engraftment levels in the peripheral blood and bonemarrow. Peak levels of chimeric antigen receptor T cells were temporallycorrelated with the tumor lysis syndrome. A blood sample obtained on day0 and a bone marrow sample obtained on day 1 had no PCR signal atbaseline. Flow-cytometric analysis of bone marrow aspirates at baseline(FIG. 14C) shows predominant infiltration with CD19+CD5+ cells that wereclonal, as assessed by means of immunoglobulin kappa light-chainstaining, with a paucity of T cells. On day 31 after infusion, CD5+ Tcells were present, and no normal or malignant B cells were detected.The numbers indicate the relative frequency of cells in each quadrant.Both the x axis and the y axis show a log 10 scale. The gating strategyinvolved an initial gating on CD19+ and CD5+ cells in the boxes on theleft, and the subsequent identification of immunoglobulin kappa andlambda expression on the CD19+CD5+ subset (boxes on the right)

DETAILED DESCRIPTION

The invention relates to compositions and methods for treating cancerincluding but not limited to hematologic malignancies and solid tumors.The present invention relates to a strategy of adoptive cell transfer ofT cells transduced to express a chimeric antigen receptor (CAR). CARsare molecules that combine antibody-based specificity for a desiredantigen (e.g., tumor antigen) with a T cell receptor-activatingintracellular domain to generate a chimeric protein that exhibits aspecific anti-tumor cellular immune activity.

The present invention relates generally to the use of T cellsgenetically modified to stably express a desired CAR. T cells expressinga CAR are referred to herein as CAR T cells or CAR modified T cells.Preferably, the cell can be genetically modified to stably express anantibody binding domain on its surface, conferring novel antigenspecificity that is MHC independent. In some instances, the T cell isgenetically modified to stably express a CAR that combines an antigenrecognition domain of a specific antibody with an intracellular domainof the CD3-zeta chain or FcγRI protein into a single chimeric protein.

In one embodiment, the CAR of the invention comprises an extracellulardomain having an antigen recognition domain, a transmembrane domain, anda cytoplasmic domain. In one embodiment, the transmembrane domain thatnaturally is associated with one of the domains in the CAR is used. Inanother embodiment, the transmembrane domain can be selected or modifiedby amino acid substitution to avoid binding of such domains to thetransmembrane domains of the same or different surface membrane proteinsto minimize interactions with other members of the receptor complex.Preferably, the transmembrane domain is the CD8α hinge domain.

With respect to the cytoplasmic domain, the CAR of the invention can bedesigned to comprise the CD28 and/or 4-1 BB signaling domain by itselfor be combined with any other desired cytoplasmic domain(s) useful inthe context of the CAR of the invention. In one embodiment, thecytoplasmic domain of the CAR can be designed to further comprise thesignaling domain of CD3-zeta. For example, the cytoplasmic domain of theCAR can include but is not limited to CD3-zeta, 4-1BB and CD28 signalingmodules and combinations thereof. Accordingly, the invention providesCAR T cells and methods of their use for adoptive therapy.

In one embodiment, the CAR T cells of the invention can be generated byintroducing a lentiviral vector comprising a desired CAR, for example aCAR comprising anti-CD19, CD8α hinge and transmembrane domain, and human4-1BB and CD3zeta signaling domains, into the cells. The CAR T cells ofthe invention are able to replicate in vivo resulting in long-termpersistence that can lead to sustained tumor control.

In one embodiment the invention relates to administering a geneticallymodified T cell expressing a CAR for the treatment of a patient havingcancer or at risk of having cancer using lymphocyte infusion.Preferably, autologous lymphocyte infusion is used in the treatment.Autologous PBMCs are collected from a patient in need of treatment and Tcells are activated and expanded using the methods described herein andknown in the art and then infused back into the patient.

In yet another embodiment, the invention relates generally to thetreatment of a patient at risk of developing CLL. The invention alsoincludes treating a malignancy or an autoimmune disease in whichchemotherapy and/or immunotherapy in a patient results in significantimmunosuppression in the patient, thereby increasing the risk of thepatient of developing CLL.

The invention includes using T cells expressing an anti-CD19 CARincluding both CD3-zeta and the 4-1BB costimulatory domain (alsoreferred to as CART19 T cells). The CART19 T cells of the invention canundergo robust in vivo T cell expansion and can establish CD19-specificmemory cells that persist at high levels for an extended amount of timein blood and bone marrow. In some instances, the CART19 T cells of theinvention infused into a patient can eliminate leukemia cells in vive inpatients with advanced chemotherapy-resistant CLL. However, theinvention is not limited to CART19 T cells. Rather, the inventionincludes any antigen binding moiety fused with one or more intracellulardomains selected from the group of a CD137 (4-1BB) signaling domain, aCD28 signaling domain, a CD3zeta signal domain, and any combinationthereof.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

“Activation”, as used herein, refers to the state of a T cell that hasbeen sufficiently stimulated to induce detectable cellularproliferation. Activation can also be associated with induced cytokineproduction, and detectable effector functions. The term “activated Tcells” refers to, among other things, T cells that are undergoing celldivision.

The term “antibody.” as used herein, refers to an immunoglobulinmolecule which specifically binds with an antigen. Antibodies can beintact immunoglobulins derived from natural sources or from recombinantsources and can be immunoreactive portions of intact immunoglobulins.Antibodies are typically tetramers of immunoglobulin molecules. Theantibodies in the present invention may exist in a variety of formsincluding, for example, polyclonal antibodies, monoclonal antibodies,Fv, Fab and F(ab)₂, as well as single chain antibodies and humanizedantibodies (Harlow et al., 1999, In: Using Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989,In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houstonet al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al.,1988, Science 242:423-426).

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limitedto, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, scFvantibodies, and multispecific antibodies formed from antibody fragments.

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations.

An “antibody light chain,” as used herein, refers to the smaller of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations. κ and λ light chains refer tothe two major antibody light chain isotypes.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to elicit thedesired immune response. Moreover, a skilled artisan will understandthat an antigen need not be encoded by a “gene” at all. It is readilyapparent that an antigen can be generated synthesized or can be derivedfrom a biological sample. Such a biological sample can include, but isnot limited to a tissue sample, a tumor sample, a cell or a biologicalfluid.

The term “anti-tumor effect” as used herein, refers to a biologicaleffect which can be manifested by a decrease in tumor volume, a decreasein the number of tumor cells, a decrease in the number of metastases, anincrease in life expectancy, or amelioration of various physiologicalsymptoms associated with the cancerous condition. An “anti-tumor effect”can also be manifested by the ability of the peptides, polynucleotides,cells and antibodies of the invention in prevention of the occurrence oftumor in the first place.

The term “auto-antigen” means, in accordance with the present invention,any self-antigen which is mistakenly recognized by the immune system asbeing foreign. Auto-antigens comprise, but are not limited to, cellularproteins, phosphoproteins, cellular surface proteins, cellular lipids,nucleic acids, glycoproteins, including cell surface receptors.

The term “autoimmune disease” as used herein is defined as a disorderthat results from an autoimmune response. An autoimmune disease is theresult of an inappropriate and excessive response to a self-antigen.Examples of autoimmune diseases include but are not limited to,Addision's disease, alopecia greata, ankylosing spondylitis, autoimmunehepatitis, autoimmune parotitis. Crohn's disease, diabetes (Type I),dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis,Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolyticanemia, systemic lupus erythematosus, multiple sclerosis, myastheniagravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome,spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema,pemicious anemia, ulcerative colitis, among others.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

“Allogeneic” refers to a graft derived from a different animal of thesame species.

“Xenogeneic” refers to a graft derived from an animal of a differentspecies.

The term “cancer” as used herein is defined as disease characterized bythe rapid and uncontrolled growth of aberrant cells. Cancer cells canspread locally or through the bloodstream and lymphatic system to otherparts of the body. Examples of various cancers include but are notlimited to, breast cancer, prostate cancer, ovarian cancer, cervicalcancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer,liver cancer, brain cancer, lymphoma, leukemia, lung cancer and thelike.

“Co-stimulatory ligand.” as the term is used herein, includes a moleculeon an antigen presenting cell (e.g., an aAPC, dendritic cell, B cell,and the like) that specifically binds a cognate co-stimulatory moleculeon a T cell, thereby providing a signal which, in addition to theprimary signal provided by, for instance, binding of a TCR/CD3 complexwith an MHC molecule loaded with peptide, mediates a T cell response,including, but not limited to, proliferation, activation,differentiation, and the like. A co-stimulatory ligand can include, butis not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L, PD-L2, 4-1BBL,OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesionmolecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G. MICA, MICB, HVEM,lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist orantibody that binds Toll ligand receptor and a ligand that specificallybinds with B7-H3. A co-stimulatory ligand also encompasses, inter alia,an antibody that specifically binds with a co-stimulatory moleculepresent on a T cell, such as, but not limited to, CD27, CD28, 4-1BB.OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specificallybinds with CD83.

A “co-stimulatory molecule” refers to the cognate binding partner on a Tcell that specifically binds with a co-stimulatory ligand, therebymediating a co-stimulatory response by the T cell, such as, but notlimited to, proliferation. Co-stimulatory molecules include, but are notlimited to an MHC class I molecule, BTLA and a Toll ligand receptor.

A “co-stimulatory signal”, as used herein, refers to a signal, which incombination with a primary signal, such as TCR/CD3 ligation, leads to Tcell proliferation and/or upregulation or downregulation of keymolecules.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate. In contrast, a “disorder”in an animal is a state of health in which the animal is able tomaintain homeostasis, but in which the animal's state of health is lessfavorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

An “effective amount” as used herein, means an amount which provides atherapeutic or prophylactic benefit.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system Expression vectors include all those known in the art,such as cosmids, plasmids (e.g., naked or contained in liposomes) andviruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

“Homologous” refers to the sequence similarity or sequence identitybetween two polypeptides or between two nucleic acid molecules. When aposition in both of the two compared sequences is occupied by the samebase or amino acid monomer subunit, e.g., if a position in each of twoDNA molecules is occupied by adenine, then the molecules are homologousat that position. The percent of homology between two sequences is afunction of the number of matching or homologous positions shared by thetwo sequences divided by the number of positions compared X 100. Forexample, if 6 of 10 of the positions in two sequences are matched orhomologous then the two sequences are 60% homologous. By way of example,the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, acomparison is made when two sequences are aligned to give maximumhomology.

The term “immunoglobulin” or “Ig,” as used herein is defined as a classof proteins, which function as antibodies. Antibodies expressed by Bcells are sometimes referred to as the BCR (B cell receptor) or antigenreceptor. The five members included in this class of proteins are IgA,IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present inbody secretions, such as saliva, tears, breast milk, gastrointestinalsecretions and mucus secretions of the respiratory and genitourinarytracts. IgG is the most common circulating antibody. IgM is the mainimmunoglobulin produced in the primary immune response in most subjects.It is the most efficient immunoglobulin in agglutination, complementfixation, and other antibody responses, and is important in defenseagainst bacteria and viruses. IgD is the immunoglobulin that has noknown antibody function, but may serve as an antigen receptor. IgE isthe immunoglobulin that mediates immediate hypersensitivity by causingrelease of mediators from mast cells and basophils upon exposure toallergen.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the compositions and methods ofthe invention. The instructional material of the kit of the inventionmay, for example, be affixed to a container which contains the nucleicacid, peptide, and/or composition of the invention or be shippedtogether with a container which contains the nucleic acid, peptide,and/or composition. Alternatively, the instructional material may beshipped separately from the container with the intention that theinstructional material and the compound be used cooperatively by therecipient.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

A “lentivirus” as used herein refers to a genus of the Retroviridaefamily. Lentiviruses are unique among the retroviruses in being able toinfect non-dividing cells; they can deliver a significant amount ofgenetic information into the DNA of the host cell, so they are one ofthe most efficient methods of a gene delivery vector. HIV, SIV, and FIVare all examples of lentiviruses. Vectors derived from lentivirusesoffer the means to achieve significant levels of gene transfer in vivo.

By the term “modulating,” as used herein, is meant mediating adetectable increase or decrease in the level of a response in a subjectcompared with the level of a response in the subject in the absence of atreatment or compound, and/or compared with the level of a response inan otherwise identical but untreated subject. The term encompassesperturbing and/or affecting a native signal or response therebymediating a beneficial therapeutic response in a subject, preferably, ahuman.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

The term “operably linked” refers to functional linkage between aregulatory sequence and a heterologous nucleic acid sequence resultingin expression of the latter. For example, a first nucleic acid sequenceis operably linked with a second nucleic acid sequence when the firstnucleic acid sequence is placed in a functional relationship with thesecond nucleic acid sequence. For instance, a promoter is operablylinked to a coding sequence if the promoter affects the transcription orexpression of the coding sequence. Generally, operably linked DNAsequences are contiguous and, where necessary to join two protein codingregions, in the same reading frame.

The term “overexpressed” tumor antigen or “overexpression” of the tumorantigen is intended to indicate an abnormal level of expression of thetumor antigen in a cell from a disease area like a solid tumor within aspecific tissue or organ of the patient relative to the level ofexpression in a normal cell from that tissue or organ. Patients havingsolid tumors or a hematological malignancy characterized byoverexpression of the tumor antigen can be determined by standard assaysknown in the art.

“Parenteral” administration of an immunogenic composition includes,e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis a human.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR™, and thelike, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

The term “promoter” as used herein is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa polynucleotide sequence.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell under most or allphysiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell substantially only whenan inducer which corresponds to the promoter is present in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide encodes or specified by a gene,causes the gene product to be produced in a cell substantially only ifthe cell is a cell of the tissue type corresponding to the promoter.

By the term “specifically binds.” as used herein with respect to anantibody, is meant an antibody which recognizes a specific antigen, butdoes not substantially recognize or bind other molecules in a sample.For example, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific. In another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific. In some instances,the terms “specific binding” or “specifically binding,” can be used inreference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, to mean that the interaction isdependent upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the chemical species; for example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

By the term “stimulation,” is meant a primary response induced bybinding of a stimulatory molecule (e.g., a TCR/CD3 complex) with itscognate ligand thereby mediating a signal transduction event, such as,but not limited to, signal transduction via the TCR/CD3 complex.Stimulation can mediate altered expression of certain molecules, such asdownregulation of TGF-β, and/or reorganization of cytoskeletalstructures, and the like.

A “stimulatory molecule,” as the term is used herein, means a moleculeon a T cell that specifically binds with a cognate stimulatory ligandpresent on an antigen presenting cell.

A “stimulatory ligand,” as used herein, means a ligand that when presenton an antigen presenting cell (e.g., an aAPC, a dendritic cell, aB-cell, and the like) can specifically bind with a cognate bindingpartner (referred to herein as a “stimulatory molecule”) on a T cell,thereby mediating a primary response by the T cell, including, but notlimited to, activation, initiation of an immune response, proliferation,and the like. Stimulatory ligands are well-known in the art andencompass, inter alia, an MHC Class I molecule loaded with a peptide, ananti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonistanti-CD2 antibody.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals). Examples of subjectsinclude humans, dogs, cats, mice, rats, and transgenic species thereof.

As used herein, a “substantially purified” cell is a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cell that have been separated from the cells with which theyare naturally associated in their natural state. In some embodiments,the cells are cultured in vitro. In other embodiments, the cells are notcultured in vitro.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

The term “therapeutically effective amount” refers to the amount of thesubject compound that will elicit the biological or medical response ofa tissue, system, or subject that is being sought by the researcher,veterinarian, medical doctor or other clinician. The term“therapeutically effective amount” includes that amount of a compoundthat, when administered, is sufficient to prevent development of, oralleviate to some extent, one or more of the signs or symptoms of thedisorder or disease being treated. The therapeutically effective amountwill vary depending on the compound, the disease and its severity andthe age, weight, etc., of the subject to be treated.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

The phrase “under transcriptional control” or “operatively linked” asused herein means that the promoter is in the correct location andorientation in relation to a polynucleotide to control the initiation oftranscription by RNA polymerase and expression of the polynucleotide.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,and the like.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

DESCRIPTION

The present invention provides compositions and methods for treatingcancer among other diseases. The cancer may be a hematologicalmalignancy, a solid tumor, a primary or a metatastizing tumor.Preferably, the cancer is a hematological malignancy, and morepreferably, the cancer is Chronic Lymphocytic Leukemia (CLL). Otherdiseases treatable using the compositions and methods of the inventioninclude viral, bacterial and parasitic infections as well as autoimmunediseases.

In one embodiment, the invention provides a cell (e.g., T cell)engineered to express a CAR wherein the CAR T cell exhibits an antitumorproperty. The CAR of the invention can be engineered to comprise anextracellular domain having an antigen binding domain fused to anintracellular signaling domain of the T cell antigen receptor complexzeta chain (e.g., CD3 zeta). The CAR of the invention when expressed ina T cell is able to redirect antigen recognition based on the antigenbinding specificity. An exemplary antigen is CD19 because this antigenis expressed on malignant B cells. However, the invention is not limitedto targeting CD19. Rather, the invention includes any antigen bindingmoiety that when bound to its cognate antigen, affects a tumor cell sothat the tumor cell fails to grow, is prompted to die, or otherwise isaffected so that the tumor burden in a patient is diminished oreliminated. The antigen binding moiety is preferably fused with anintracellular domain from one or more of a costimulatory molecule and azeta chain. Preferably, the antigen binding moiety is fused with one ormore intracellular domains selected from the group of a CD137 (4-1BB)signaling domain, a CD28 signaling domain, a CD3zeta signal domain, andany combination thereof.

In one embodiment, the CAR of the invention comprises a CD137 (4-1BB)signaling domain. This is because the present invention is partly basedon the discovery that CAR-mediated T-cell responses can be furtherenhanced with the addition of costimulatory domains. For example,inclusion of the CD137 (4-1BB) signaling domain significantly increasedanti-tumor activity and in vivo persistence of CAR T cells compared toan otherwise identical CAR T cell not engineered to express CD137(4-1BB).

Composition

The present invention provides chimeric antigen receptor (CAR)comprising an extracellular and intracellular domain. The extracellulardomain comprises a target-specific binding element otherwise referred toas an antigen binding moiety. The intracellular domain or otherwise thecytoplasmic domain comprises, a costimulatory signaling region and azeta chain portion. The costimulatory signaling region refers to aportion of the CAR comprising the intracellular domain of acostimulatory molecule. Costimulatory molecules are cell surfacemolecules other than antigens receptors or their ligands that arerequired for an efficient response of lymphocytes to antigen.

Between the extracellular domain and the transmembrane domain of theCAR, or between the cytoplasmic domain and the transmembrane domain ofthe CAR, there may be incorporated a spacer domain. As used herein, theterm “spacer domain” generally means any oligo- or polypeptide thatfunctions to link the transmembrane domain to, either the extracellulardomain or, the cytoplasmic domain in the polypeptide chain. A spacerdomain may comprise up to 300 amino acids, preferably 10 to 100 aminoacids and most preferably 25 to 50 amino acids.

Antigen Bindine Moiety

In one embodiment, the CAR of the invention comprises a target-specificbinding element otherwise referred to as an antigen binding moiety. Thechoice of moiety depends upon the type and number of ligands that definethe surface of a target cell. For example, the antigen binding domainmay be chosen to recognize a ligand that acts as a cell surface markeron target cells associated with a particular disease state. Thusexamples of cell surface markers that may act as ligands for the antigenmoiety domain in the CAR of the invention include those associated withviral, bacterial and parasitic infections, autoimmune disease and cancercells.

In one embodiment, the CAR of the invention can be engineered to targeta tumor antigen of interest by way of engineering a desired antigenbinding moiety that specifically binds to an antigen on a tumor cell. Inthe context of the present invention, “tumor antigen” or“hyperporoliferative disorder antigen” or “antigen associated with ahyperproliferative disorder,” refers to antigens that are common tospecific hyperproliferative disorders such as cancer. The antigensdiscussed herein are merely included by way of example. The list is notintended to be exclusive and further examples will be readily apparentto those of skill in the art.

Tumor antigens are proteins that are produced by tumor cells that elicitan immune response, particularly T-cell mediated immune responses. Theselection of the antigen binding moiety of the invention will depend onthe particular type of cancer to be treated. Tumor antigens are wellknown in the art and include, for example, a glioma-associated antigen,carcinoembryonic antigen (CEA), β-human chorionic gonadotropin,alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1,MN-CA IX, human telomerase reverse transcriptase. RU1, RU2 (AS),intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase,prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein,PSMA, Her2/neu, survivin and telomerase, prostate-carcinoma tumorantigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22,insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

In one embodiment, the tumor antigen comprises one or more antigeniccancer epitopes associated with a malignant tumor. Malignant tumorsexpress a number of proteins that can serve as target antigens for animmune attack. These molecules include but are not limited totissue-specific antigens such as MART-1, tyrosinase and GP 100 inmelanoma and prostatic acid phosphatase (PAP) and prostate-specificantigen (PSA) in prostate cancer. Other target molecules belong to thegroup of transformation-related molecules such as the oncogeneHER-2/Neu/ErbB-2. Yet another group of target antigens are onco-fetalantigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma thetumor-specific idiotype immunoglobulin constitutes a trulytumor-specific immunoglobulin antigen that is unique to the individualtumor. B-cell differentiation antigens such as CD19, CD20 and CD37 areother candidates for target antigens in B-cell lymphoma. Some of theseantigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targetsfor passive immunotherapy with monoclonal antibodies with limitedsuccess.

The type of tumor antigen referred to in the invention may also be atumor-specific antigen (TSA) or a tumor-associated antigen (TAA). A TSAis unique to tumor cells and does not occur on other cells in the body.A TAA associated antigen is not unique to a tumor cell and instead isalso expressed on a normal cell under conditions that fail to induce astate of immunologic tolerance to the antigen. The expression of theantigen on the tumor may occur under conditions that enable the immunesystem to respond to the antigen. TAAs may be antigens that areexpressed on normal cells during fetal development when the immunesystem is immature and unable to respond or they may be antigens thatare normally present at extremely low levels on normal cells but whichare expressed at much higher levels on tumor cells.

Non-limiting examples of TSA or TAA antigens include the following:Differentiation antigens such as MART-1/MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigenssuch as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressedembryonic antigens such as CEA; overexpressed oncogenes and mutatedtumor-suppressor genes such as p53, Ras, HER-2/neu; unique tumorantigens resulting from chromosomal translocations; such as BCR-ABL,E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as theEpstein Barr virus antigens EBVA and the human papillomavirus (HPV)antigens E6 and E7. Other large, protein-based antigens include TSP-180,MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met,nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras,beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72,alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250,Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1,RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associatedprotein, TAAL6, TAG72, TLP, and TPS.

In a preferred embodiment, the antigen binding moiety portion of the CARtargets an antigen that includes but is not limited to CD19, CD20, CD22,ROR1, Mesothelin, CD33/IL3Ra, c-Met, PSMA. Glycolipid F77, EGFRvIII,GD-2, MY-ESO-1 TCR, MAGE A3 TCR, and the like.

Depending on the desired antigen to be targeted, the CAR of theinvention can be engineered to include the appropriate antigen bindmoiety that is specific to the desired antigen target. For example, ifCD19 is the desired antigen that is to be targeted, an antibody for CD19can be used as the antigen bind moiety for incorporation into the CAR ofthe invention.

In one embodiment, the antigen binding moiety portion of the CAR of theinvention targets CD19. Preferably, the antigen binding moiety portionin the CAR of the invention is anti-CD19 scFV, wherein the nucleic acidsequence of the anti-CD19 scFV comprises the sequence set forth in SEQID: 14. In one embodiment, the anti-CD19 scFV comprise the nucleic acidsequence that encodes the amino acid sequence of SEQ ID NO: 20. Inanother embodiment, the anti-CD19 scFV portion of the CAR of theinvention comprises the amino acid sequence set forth in SEQ ID NO: 20.

Transmembrane Domain

With respect to the transmembrane domain, the CAR can be designed tocomprise a transmembrane domain that is fused to the extracellulardomain of the CAR. In one embodiment, the transmembrane domain thatnaturally is associated with one of the domains in the CAR is used. Insome instances, the transmembrane domain can be selected or modified byamino acid substitution to avoid binding of such domains to thetransmembrane domains of the same or different surface membrane proteinsto minimize interactions with other members of the receptor complex.

The transmembrane domain may be derived either from a natural or from asynthetic source. Where the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein. Transmembrane regionsof particular use in this invention may be derived from (i.e. compriseat least the transmembrane region(s) of) the alpha, beta or zeta chainof the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9,CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.Alternatively the transmembrane domain may be synthetic, in which caseit will comprise predominantly hydrophobic residues such as leucine andvaline. Preferably a triplet of phenylalanine, tryptophan and valinewill be found at each end of a synthetic transmembrane domain.Optionally, a short oligo- or polypeptide linker, preferably between 2and 10 amino acids in length may form the linkage between thetransmembrane domain and the cytoplasmic signaling domain of the CAR. Aglycine-serine doublet provides a particularly suitable linker.

Preferably, the transmembrane domain in the CAR of the invention is theCD8 transmembrane domain. In one embodiment, the CD8 transmembranedomain comprises the nucleic acid sequence of SEQ ID NO: 16. In oneembodiment, the CD8 transmembrane domain comprises the nucleic acidsequence that encodes the amino acid sequence of SEQ ID NO: 22. Inanother embodiment, the CD8 transmembrane domain comprises the aminoacid sequence of SEQ ID NO: 22.

In some instances, the transmembrane domain of the CAR of the inventioncomprises the CD8α hinge domain. In one embodiment, the CD8 hinge domaincomprises the nucleic acid sequence of SEQ ID NO: 15. In one embodiment,the CD8 hinge domain comprises the nucleic acid sequence that encodesthe amino acid sequence of SEQ ID NO: 21. In another embodiment, the CD8hinge domain comprises the amino acid sequence of SEQ ID NO: 21.

Cytoplasmic Domain

The cytoplasmic domain or otherwise the intracellular signaling domainof the CAR of the invention is responsible for activation of at leastone of the normal effector functions of the immune cell in which the CARhas been placed in. The term “effector function” refers to a specializedfunction of a cell. Effector function of a T cell, for example, may becytolytic activity or helper activity including the secretion ofcytokines. Thus the term “intracellular signaling domain” refers to theportion of a protein which transduces the effector function signal anddirects the cell to perform a specialized function. While usually theentire intracellular signaling domain can be employed, in many cases itis not necessary to use the entire chain. To the extent that a truncatedportion of the intracellular signaling domain is used, such truncatedportion may be used in place of the intact chain as long as ittransduces the effector function signal. The term intracellularsignaling domain is thus meant to include any truncated portion of theintracellular signaling domain sufficient to transduce the effectorfunction signal.

Preferred examples of intracellular signaling domains for use in the CARof the invention include the cytoplasmic sequences of the T cellreceptor (TCR) and co-receptors that act in concert to initiate signaltransduction following antigen receptor engagement, as well as anyderivative or variant of these sequences and any synthetic sequence thathas the same functional capability.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondary orco-stimulatory signal is also required. Thus, T cell activation can besaid to be mediated by two distinct classes of cytoplasmic signalingsequence: those that initiate antigen-dependent primary activationthrough the TCR (primary cytoplasmic signaling sequences) and those thatact in an antigen-independent manner to provide a secondary orco-stimulatory signal (secondary cytoplasmic signaling sequences).

Primary cytoplasmic signaling sequences regulate primary activation ofthe TCR complex either in a stimulatory way, or in an inhibitory way.Primary cytoplasmic signaling sequences that act in a stimulatory mannermay contain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs.

Examples of ITAM containing primary cytoplasmic signaling sequences thatare of particular use in the invention include those derived from TCRzeta. FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22,CD79a, CD79b, and CD66d. It is particularly preferred that cytoplasmicsignaling molecule in the CAR of the invention comprises a cytoplasmicsignaling sequence derived from CD3 zeta.

In a preferred embodiment, the cytoplasmic domain of the CAR can bedesigned to comprise the CD3-zeta signaling domain by itself or combinedwith any other desired cytoplasmic domain(s) useful in the context ofthe CAR of the invention. For example, the cytoplasmic domain of the CARcan comprise a CD3 zeta chain portion and a costimulatory signalingregion. The costimulatory signaling region refers to a portion of theCAR comprising the intracellular domain of a costimulatory molecule. Acostimulatory molecule is a cell surface molecule other than an antigenreceptor or their ligands that is required for an efficient response oflymphocytes to an antigen. Examples of such molecules include CD27,CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C. B7-H3,and a ligand that specifically binds with CD83, and the like. Thus,while the invention in exemplified primarily with 4-1BB as theco-stimulatory signaling element, other costimulatory elements arewithin the scope of the invention.

The cytoplasmic signaling sequences within the cytoplasmic signalingportion of the CAR of the invention may be linked to each other in arandom or specified order. Optionally, a short oligo- or polypeptidelinker, preferably between 2 and 10 amino acids in length may form thelinkage. A glycine-serine doublet provides a particularly suitablelinker.

In one embodiment, the cytoplasmic domain is designed to comprise thesignaling domain of CD3-zeta and the signaling domain of CD28. Inanother embodiment, the cytoplasmic domain is designed to comprise thesignaling domain of CD3-zeta and the signaling domain of 4-1BB. In yetanother embodiment, the cytoplasmic domain is designed to comprise thesignaling domain of CD3-zeta and the signaling domain of CD28 and 4-1BB.

In one embodiment, the cytoplasmic domain in the CAR of the invention isdesigned to comprise the signaling domain of 4-1BB and the signalingdomain of CD3-zeta, wherein the signaling domain of 4-1BB comprises thenucleic acid sequence set forth in SEQ ID NO: 17 and the signalingdomain of CD3-zeta comprises the nucleic acid sequence set forth in SEQID NO: 18.

In one embodiment, the cytoplasmic domain in the CAR of the invention isdesigned to comprise the signaling domain of 4-1BB and the signalingdomain of CD3-zeta, wherein the signaling domain of 4-1BB comprises thenucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:23 and the signaling domain of CD3-zeta comprises the nucleic acidsequence that encodes the amino acid sequence of SEQ ID NO: 24.

In one embodiment, the cytoplasmic domain in the CAR of the invention isdesigned to comprise the signaling domain of 4-1BB and the signalingdomain of CD3-zeta, wherein the signaling domain of 4-1BB comprises theamino acid sequence set forth in SEQ ID NO: 23 and the signaling domainof CD3-zeta comprises the amino acid sequence set forth in SEQ ID NO:24.

Vectors

The present invention encompasses a DNA construct comprising sequencesof a CAR, wherein the sequence comprises the nucleic acid sequence of anantigen binding moiety operably linked to the nucleic acid sequence ofan intracellular domain. An exemplary intracellular domain that can beused in the CAR of the invention includes but is not limited to theintracellular domain of CD3-zeta, CD28, 4-1BB, and the like. In someinstances, the CAR can comprise any combination of CD3-zeta, CD28,4-1BB, and the like.

In one embodiment, the CAR of the invention comprises anti-CD19 scFv,human CD8 hinge and transmembrane domain, and human 4-1BB and CD3zetasignaling domains. In one embodiment, the CAR of the invention comprisesthe nucleic acid sequence set forth in SEQ ID NO: 8. In anotherembodiment, the CAR of the invention comprises the nucleic acid sequencethat encodes the amino acid sequence of SEQ ID NO: 12. In anotherembodiment, the CAR of the invention comprises the amino acid sequenceset forth in SEQ ID NO: 12.

The nucleic acid sequences coding for the desired molecules can beobtained using recombinant methods known in the art, such as, forexample by screening libraries from cells expressing the gene, byderiving the gene from a vector known to include the same, or byisolating directly from cells and tissues containing the same, usingstandard techniques. Alternatively, the gene of interest can be producedsynthetically, rather than cloned.

The present invention also provides vectors in which a DNA of thepresent invention is inserted. Vectors derived from retroviruses such asthe lentivirus are suitable tools to achieve long-term gene transfersince they allow long-term, stable integration of a transgene and itspropagation in daughter cells. Lentiviral vectors have the addedadvantage over vectors derived from onco-retroviruses such as murineleukemia viruses in that they can transduce non-proliferating cells,such as hepatocytes. They also have the added advantage of lowimmunogenicity.

In brief summary, the expression of natural or synthetic nucleic acidsencoding CARs is typically achieved by operably linking a nucleic acidencoding the CAR polypeptide or portions thereof to a promoter, andincorporating the construct into an expression vector. The vectors canbe suitable for replication and integration eukaryotes. Typical cloningvectors contain transcription and translation terminators, initiationsequences, and promoters useful for regulation of the expression of thedesired nucleic acid sequence.

The expression constructs of the present invention may also be used fornucleic acid immunization and gene therapy, using standard gene deliveryprotocols. Methods for gene delivery are known in the art. See, e.g.,U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589.466, incorporated byreference herein in their entireties. In another embodiment, theinvention provides a gene therapy vector.

The nucleic acid can be cloned into a number of types of vectors. Forexample, the nucleic acid can be cloned into a vector including, but notlimited to a plasmid, a phagemid, a phage derivative, an animal virus,and a cosmid. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al. (2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers, (e.g., WO01/%96584: WO 01/29058; and U.S. Pat. No. 6,326,193).

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, adenovirus vectorsare used. A number of adenovirus vectors are known in the art. In oneembodiment, lentivirus vectors are used.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Growth Factor-1α(EF-1α). However, other constitutive promoter sequences may also beused, including, but not limited to the simian virus 40 (SV40) earlypromoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avianleukemia virus promoter, an Epstein-Barr virus immediate early promoter,a Rous sarcoma virus promoter, as well as human gene promoters such as,but not limited to, the actin promoter, the myosin promoter, thehemoglobin promoter, and the creatine kinase promoter. Further, theinvention should not be limited to the use of constitutive promoters.Inducible promoters are also contemplated as part of the invention. Theuse of an inducible promoter provides a molecular switch capable ofturning on expression of the polynucleotide sequence which it isoperatively linked when such expression is desired, or turning off theexpression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

In order to assess the expression of a CAR polypeptide or portionsthereof, the expression vector to be introduced into a cell can alsocontain either a selectable marker gene or a reporter gene or both tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In other aspects, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, for example, antibiotic-resistance genes,such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York). A preferred method for the introduction of a polynucleotideinto a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the recombinant DNAsequence in the host cell, a variety of assays may be performed. Suchassays include, for example, “molecular biological” assays well known tothose of skill in the art, such as Southern and Northern blotting,RT-PCR and PCR; “biochemical” assays, such as detecting the presence orabsence of a particular peptide, e.g., by immunological means (ELISAsand Western blots) or by assays described herein to identify agentsfalling within the scope of the invention.

Sources of T Cells

Prior to expansion and genetic modification of the T cells of theinvention, a source of T cells is obtained from a subject. T cells canbe obtained from a number of sources, including peripheral bloodmononuclear cells, bone marrow, lymph node tissue, cord blood, thymustissue, tissue from a site of infection, ascites, pleural effusion,spleen tissue, and tumors. In certain embodiments of the presentinvention, any number of T cell lines available in the art, may be used.In certain embodiments of the present invention, T cells can be obtainedfrom a unit of blood collected from a subject using any number oftechniques known to the skilled artisan, such as Ficoll™ separation. Inone preferred embodiment, cells from the circulating blood of anindividual are obtained by apheresis. The apheresis product typicallycontains lymphocytes, including T cells, monocytes, granulocytes, Bcells, other nucleated white blood cells, red blood cells, andplatelets. In one embodiment, the cells collected by apheresis may bewashed to remove the plasma fraction and to place the cells in anappropriate buffer or media for subsequent processing steps. In oneembodiment of the invention, the cells are washed with phosphatebuffered saline (PBS). In an alternative embodiment, the wash solutionlacks calcium and may lack magnesium or may lack many if not alldivalent cations. Again, surprisingly, initial activation steps in theabsence of calcium lead to magnified activation. As those of ordinaryskill in the art would readily appreciate a washing step may beaccomplished by methods known to those in the art, such as by using asemi-automated “flow-through” centrifuge (for example, the Cobe 2991cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5)according to the manufacturer's instructions. After washing, the cellsmay be resuspended in a variety of biocompatible buffers, such as, forexample, Ca²⁺-free, Mg²⁺-free PBS, PlasmaLyte A, or other salinesolution with or without buffer. Alternatively, the undesirablecomponents of the apheresis sample may be removed and the cells directlyresuspended in culture media.

In another embodiment, T cells are isolated from peripheral bloodlymphocytes by lysing the red blood cells and depleting the monocytes,for example, by centrifugation through a PERCOLL™ gradient or bycounterflow centrifugal elutriation. A specific subpopulation of Tcells, such as CD³⁺, CD28⁺, CD4⁺, CD8⁺, CD45RA⁺, and CD45RO⁺ T cells,can be further isolated by positive or negative selection techniques.For example, in one embodiment, T cells are isolated by incubation withanti-CD3/anti-CD28 (i.e., 3×28)-conjugated beads, such as DYNABEADS®M-450 CD3/CD28 T, for a time period sufficient for positive selection ofthe desired T cells. In one embodiment, the time period is about 30minutes. In a further embodiment, the time period ranges from 30 minutesto 36 hours or longer and all integer values there between. In a furtherembodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. Inyet another preferred embodiment, the time period is 10 to 24 hours. Inone preferred embodiment, the incubation time period is 24 hours. Forisolation of T cells from patients with leukemia, use of longerincubation times, such as 24 hours, can increase cell yield. Longerincubation times may be used to isolate T cells in any situation wherethere are few T cells as compared to other cell types, such in isolatingtumor infiltrating lymphocytes (TIL) from tumor tissue or fromimmune-compromised individuals. Further, use of longer incubation timescan increase the efficiency of capture of CD8+ T cells. Thus, by simplyshortening or lengthening the time T cells are allowed to bind to theCD3/CD28 beads and/or by increasing or decreasing the ratio of beads toT cells (as described further herein), subpopulations of T cells can bepreferentially selected for or against at culture initiation or at othertime points during the process. Additionally, by increasing ordecreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on thebeads or other surface, subpopulations of T cells can be preferentiallyselected for or against at culture initiation or at other desired timepoints. The skilled artisan would recognize that multiple rounds ofselection can also be used in the context of this invention. In certainembodiments, it may be desirable to perform the selection procedure anduse the “unselected” cells in the activation and expansion process.“Unselected” cells can also be subjected to further rounds of selection.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. For example,to enrich for CD⁴⁺ cells by negative selection, a monoclonal antibodycocktail typically includes antibodies to CD14, CD20, CD11b, CD16,HLA-DR, and CD8. In certain embodiments, it may be desirable to enrichfor or positively select for regulatory T cells which typically expressCD4⁺, CD25⁺, CD62L^(hi), GITR⁺, and FoxP3⁺. Alternatively, in certainembodiments, T regulatory cells are depleted by anti-C25 conjugatedbeads or other similar method of selection.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain embodiments, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one embodiment, aconcentration of 2 billion cells/ml is used. In one embodiment, aconcentration of 1 billion cells/ml is used. In a further embodiment,greater than 100 million cells/ml is used. In a further embodiment, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet another embodiment, a concentration of cellsfrom 75, 80, 85, 90, 95, or 100 million cells/ml is used. In furtherembodiments, concentrations of 125 or 150 million cells/ml can be used.Using high concentrations can result in increased cell yield, cellactivation, and cell expansion. Further, use of high cell concentrationsallows more efficient capture of cells that may weakly express targetantigens of interest, such as CD28-negative T cells, or from sampleswhere there are many tumor cells present (i.e., leukemic blood, tumortissue, etc.). Such populations of cells may have therapeutic value andwould be desirable to obtain. For example, using high concentration ofcells allows more efficient selection of CD8⁺ T cells that normally haveweaker CD28 expression.

In a related embodiment, it may be desirable to use lower concentrationsof cells. By significantly diluting the mixture of T cells and surface(e.g., particles such as beads), interactions between the particles andcells is minimized. This selects for cells that express high amounts ofdesired antigens to be bound to the particles. For example, CD4⁺ T cellsexpress higher levels of CD28 and are more efficiently captured thanCD8⁺ T cells in dilute concentrations. In one embodiment, theconcentration of cells used is 5×10⁶/ml. In other embodiments, theconcentration used can be from about 1×10⁵/ml to 1×10⁶/ml, and anyinteger value in between.

In other embodiments, the cells may be incubated on a rotator forvarying lengths of time at varying speeds at either 2-10° C. or at roomtemperature.

T cells for stimulation can also be frozen after a washing step. Wishingnot to be bound by theory, the freeze and subsequent thaw step providesa more uniform product by removing granulocytes and to some extentmonocytes in the cell population. After the washing step that removesplasma and platelets, the cells may be suspended in a freezing solution.While many freezing solutions and parameters are known in the art andwill be useful in this context, one method involves using PBS containing20% DMSO and 8% human serum albumin, or culture media containing 10%Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitablecell freezing media containing for example, Hespan and PlasmaLyte A, thecells then are frozen to −80° C. at a rate of 1° per minute and storedin the vapor phase of a liquid nitrogen storage tank. Other methods ofcontrolled freezing may be used as well as uncontrolled freezingimmediately at −20° C. or in liquid nitrogen.

In certain embodiments, cryopreserved cells are thawed and washed asdescribed herein and allowed to rest for one hour at room temperatureprior to activation using the methods of the present invention.

Also contemplated in the context of the invention is the collection ofblood samples or apheresis product from a subject at a time period priorto when the expanded cells as described herein might be needed. As such,the source of the cells to be expanded can be collected at any timepoint necessary, and desired cells, such as T cells, isolated and frozenfor later use in T cell therapy for any number of diseases or conditionsthat would benefit from T cell therapy, such as those described herein.In one embodiment a blood sample or an apheresis is taken from agenerally healthy subject. In certain embodiments, a blood sample or anapheresis is taken from a generally healthy subject who is at risk ofdeveloping a disease, but who has not yet developed a disease, and thecells of interest are isolated and frozen for later use. In certainembodiments, the T cells may be expanded, frozen, and used at a latertime. In certain embodiments, samples are collected from a patientshortly after diagnosis of a particular disease as described herein butprior to any treatments. In a further embodiment, the cells are isolatedfrom a blood sample or an apheresis from a subject prior to any numberof relevant treatment modalities, including but not limited to treatmentwith agents such as natalizumab, efalizumab, antiviral agents,chemotherapy, radiation, immunosuppressive agents, such as cyclosporin,azathioprine, methotrexate, mycophenolate, and FK506, antibodies, orother immunoablative agents such as CAMPATH, anti-CD3 antibodies,cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid,steroids, FR901228, and irradiation. These drugs inhibit either thecalcium dependent phosphatase calcineurin (cyclosporine and FK506) orinhibit the p70S6 kinase that is important for growth factor inducedsignaling (rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson etal., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun.5:763-773, 1993). In a further embodiment, the cells are isolated for apatient and frozen for later use in conjunction with (e.g., before,simultaneously or following) bone marrow or stem cell transplantation, Tcell ablative therapy using either chemotherapy agents such as,fludarabine, extemal-beam radiation therapy (XRT), cyclophosphamide, orantibodies such as OKT3 or CAMPATH. In another embodiment, the cells areisolated prior to and can be frozen for later use for treatmentfollowing B-cell ablative therapy such as agents that react with CD20,e.g., Rituxan.

In a further embodiment of the present invention. T cells are obtainedfrom a patient directly following treatment. In this regard, it has beenobserved that following certain cancer treatments, in particulartreatments with drugs that damage the immune system, shortly aftertreatment during the period when patients would normally be recoveringfrom the treatment, the quality of T cells obtained may be optimal orimproved for their ability to expand ex vivo. Likewise, following exvivo manipulation using the methods described herein, these cells may bein a preferred state for enhanced engraftment and in vivo expansion.Thus, it is contemplated within the context of the present invention tocollect blood cells, including T cells, dendritic cells, or other cellsof the hematopoietic lineage, during this recovery phase. Further, incertain embodiments, mobilization (for example, mobilization withGM-CSF) and conditioning regimens can be used to create a condition in asubject wherein repopulation, recirculation, regeneration, and/orexpansion of particular cell types is favored, especially during adefined window of time following therapy. Illustrative cell typesinclude T cells, B cells, dendritic cells, and other cells of the immunesystem.

Activation and Expansion of T Cells

Whether prior to or after genetic modification of the T cells to expressa desirable CAR, the T cells can be activated and expanded generallyusing methods as described, for example, in U.S. Pat. Nos. 6,352,694;6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681;7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223;6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application PublicationNo. 20060121005.

Generally, the T cells of the invention are expanded by contact with asurface having attached thereto an agent that stimulates a CD3TCRcomplex associated signal and a ligand that stimulates a co-stimulatorymolecule on the surface of the T cells. In particular, T cellpopulations may be stimulated as described herein, such as by contactwith an anti-CD3 antibody, or antigen-binding fragment thereof, or ananti-CD2 antibody immobilized on a surface, or by contact with a proteinkinase C activator (e.g., bryostatin) in conjunction with a calciumionophore. For co-stimulation of an accessory molecule on the surface ofthe T cells, a ligand that binds the accessory molecule is used. Forexample, a population of T cells can be contacted with an anti-CD3antibody and an anti-CD28 antibody, under conditions appropriate forstimulating proliferation of the T cells. To stimulate proliferation ofeither CD4⁺ T cells or CD8⁺ T cells, an anti-CD3 antibody and ananti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3,XR-CD28 (Diaclone, Besancon, France) can be used as can other methodscommonly known in the art (Berg et al., Transplant Proc.30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328,1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

In certain embodiments, the primary stimulatory signal and theco-stimulatory signal for the T cell may be provided by differentprotocols. For example, the agents providing each signal may be insolution or coupled to a surface. When coupled to a surface, the agentsmay be coupled to the same surface (i.e., in “cis” formation) or toseparate surfaces (i.e., in “trans” formation). Alternatively, one agentmay be coupled to a surface and the other agent in solution. In oneembodiment, the agent providing the co-stimulatory signal is bound to acell surface and the agent providing the primary activation signal is insolution or coupled to a surface. In certain embodiments, both agentscan be in solution. In another embodiment, the agents may be in solubleform, and then cross-linked to a surface, such as a cell expressing Fcreceptors or an antibody or other binding agent which will bind to theagents. In this regard, see for example, U.S. Patent ApplicationPublication Nos. 20040101519 and 20060034810 for artificial antigenpresenting cells (aAPCs) that are contemplated for use in activating andexpanding T cells in the present invention.

In one embodiment, the two agents are immobilized on beads, either onthe same bead, i.e., “cis,” or to separate beads, i.e., “trans.” By wasof example, the agent providing the primary activation signal is ananti-CD3 antibody or an antigen-binding fragment thereof and the agentproviding the co-stimulatory signal is an anti-CD28 antibody orantigen-binding fragment thereof; and both agents are co-immobilized tothe same bead in equivalent molecular amounts. In one embodiment, a 1:1ratio of each antibody bound to the beads for CD4⁺ T cell expansion andT cell growth is used. In certain aspects of the present invention, aratio of anti CD3:CD28 antibodies bound to the beads is used such thatan increase in T cell expansion is observed as compared to the expansionobserved using a ratio of 1:1. In one particular embodiment an increaseof from about 1 to about 3 fold is observed as compared to the expansionobserved using a ratio of 1:1. In one embodiment, the ratio of CD3:CD28antibody bound to the beads ranges from 100:1 to 1:100 and all integervalues there between. In one aspect of the present invention, moreanti-CD28 antibody is bound to the particles than anti-CD3 antibody,i.e., the ratio of CD3:CD28 is less than one. In certain embodiments ofthe invention, the ratio of anti CD28 antibody to anti CD3 antibodybound to the beads is greater than 2:1. In one particular embodiment, a1:100 CD3:CD28 ratio of antibody bound to beads is used. In anotherembodiment, a 1:75 CD3:CD28 ratio of antibody bound to beads is used. Ina further embodiment, a 1:50 CD3:CD28 ratio of antibody bound to beadsis used. In another embodiment, a 1:30 CD3:CD28 ratio of antibody boundto beads is used. In one preferred embodiment, a 1:10 CD3:CD28 ratio ofantibody bound to beads is used. In another embodiment, a 1:3 CD3:CD28ratio of antibody bound to the beads is used. In yet another embodiment,a 3:1 CD3:CD28 ratio of antibody bound to the beads is used.

Ratios of particles to cells from 1:500 to 500:1 and any integer valuesin between may be used to stimulate T cells or other target cells. Asthose of ordinary skill in the art can readily appreciate, the ratio ofparticles to cells may depend on particle size relative to the targetcell. For example, small sized beads could only bind a few cells, whilelarger beads could bind many. In certain embodiments the ratio of cellsto particles ranges from 1:100 to 100:1 and any integer valuesin-between and in further embodiments the ratio comprises 1:9 to 9:1 andany integer values in between, can also be used to stimulate T cells.The ratio of anti-CD3- and anti-CD28-coupled particles to T cells thatresult in T cell stimulation can vary as noted above, however certainpreferred values include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8,1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, and 15:1 with one preferred ratio being at least 1:1particles per T cell. In one embodiment, a ratio of particles to cellsof 1:1 or less is used. In one particular embodiment, a preferredparticle: cell ratio is 1:5. In further embodiments, the ratio ofparticles to cells can be varied depending on the day of stimulation.For example, in one embodiment, the ratio of particles to cells is from1:1 to 10:1 on the first day and additional particles are added to thecells every day or every other day thereafter for up to 10 days, atfinal ratios of from 1:1 to 1:10 (based on cell counts on the day ofaddition). In one particular embodiment, the ratio of particles to cellsis 1:1 on the first day of stimulation and adjusted to 1:5 on the thirdand fifth days of stimulation. In another embodiment, particles areadded on a daily or every other day basis to a final ratio of 1:1 on thefirst day, and 1:5 on the third and fifth days of stimulation. Inanother embodiment, the ratio of particles to cells is 2:1 on the firstday of stimulation and adjusted to 1:10 on the third and fifth days ofstimulation. In another embodiment, particles are added on a daily orevery other day basis to a final ratio of 1:1 on the first day, and 1:10on the third and fifth days of stimulation. One of skill in the art willappreciate that a variety of other ratios may be suitable for use in thepresent invention. In particular, ratios will vary depending on particlesize and on cell size and type.

In further embodiments of the present invention, the cells, such as Tcells, are combined with agent-coated beads, the beads and the cells aresubsequently separated, and then the cells are cultured. In analternative embodiment, prior to culture, the agent-coated beads andcells are not separated but are cultured together. In a furtherembodiment, the beads and cells are first concentrated by application ofa force, such as a magnetic force, resulting in increased ligation ofcell surface markers, thereby inducing cell stimulation.

By way of example, cell surface proteins may be ligated by allowingparamagnetic beads to which anti-CD3 and anti-CD28 are attached (3×28beads) to contact the T cells. In one embodiment the cells (for example,10⁴ to 10⁹ T cells) and beads (for example, DYNABEADS® M-450 CD3/CD28 Tparamagnetic beads at a ratio of 1:1) are combined in a buffer,preferably PBS (without divalent cations such as, calcium andmagnesium). Again, those of ordinary skill in the art can readilyappreciate any cell concentration may be used. For example, the targetcell may be very rare in the sample and comprise only 0.01% of thesample or the entire sample (i.e., 100%) may comprise the target cell ofinterest. Accordingly, any cell number is within the context of thepresent invention. In certain embodiments, it may be desirable tosignificantly decrease the volume in which particles and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and particles. For example, in one embodiment, aconcentration of about 2 billion cells/ml is used. In anotherembodiment, greater than 100 million cells/ml is used. In a furtherembodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45,or 50 million cells/ml is used. In yet another embodiment, aconcentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mlis used. In further embodiments, concentrations of 125 or 150 millioncells/ml can be used. Using high concentrations can result in increasedcell yield, cell activation, and cell expansion. Further, use of highcell concentrations allows more efficient capture of cells that mayweakly express target antigens of interest, such as CD28-negative Tcells. Such populations of cells may have therapeutic value and would bedesirable to obtain in certain embodiments. For example, using highconcentration of cells allows more efficient selection of CD8+ T cellsthat normally have weaker CD28 expression.

In one embodiment of the present invention, the mixture may be culturedfor several hours (about 3 hours) to about 14 days or any hourly integervalue in between. In another embodiment, the mixture may be cultured for21 days. In one embodiment of the invention the beads and the T cellsare cultured together for about eight days. In another embodiment, thebeads and T cells are cultured together for 2-3 days. Several cycles ofstimulation may also be desired such that culture time of T cells can be60 days or more. Conditions appropriate for T cell culture include anappropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or,X-vivo 15, (Lonza)) that may contain factors necessary for proliferationand viability, including serum (e.g., fetal bovine or human serum),interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12,IL-15, TGFβ, and TNF-α or any other additives for the growth of cellsknown to the skilled artisan. Other additives for the growth of cellsinclude, but are not limited to, surfactant, plasmanate, and reducingagents such as N-acetyl-cysteine and 2-mercaptoethanol. Media caninclude RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo20. Optimizer, with added amino acids, sodium pyruvate, and vitamins,either serum-free or supplemented with an appropriate amount of serum(or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO₂).

T cells that have been exposed to varied stimulation times may exhibitdifferent characteristics. For example, typical blood or apheresedperipheral blood mononuclear cell products have a helper T cellpopulation (T_(H), CD4⁺) that is greater than the cytotoxic orsuppressor T cell population (T_(C), CD8⁺). Ex vivo expansion of T cellsby stimulating CD3 and CD28 receptors produces a population of T cellsthat prior to about days 8-9 consists predominately of T_(H) cells,while after about days 8-9, the population of T cells comprises anincreasingly greater population of T_(C) cells. Accordingly, dependingon the purpose of treatment, infusing a subject with a T cell populationcomprising predominately of T_(H) cells may be advantageous. Similarly,if an antigen-specific subset of T_(C) cells has been isolated it may bebeneficial to expand this subset to a greater degree.

Further, in addition to CD4 and CD8 markers, other phenotypic markersvary significantly, but in large part, reproducibly during the course ofthe cell expansion process. Thus, such reproducibility enables theability to tailor an activated T cell product for specific purposes.

Therapeutic Application

The present invention encompasses a cell (e.g., T cell) transduced witha lentiviral vector (LV). For example, the LV encodes a CAR thatcombines an antigen recognition domain of a specific antibody with anintracellular domain of CD3-zeta, CD28, 4-1BB, or any combinationsthereof. Therefore, in some instances, the transduced T cell can elicita CAR-mediated T-cell response.

The invention provides the use of a CAR to redirect the specificity of aprimary T cell to a tumor antigen. Thus, the present invention alsoprovides a method for stimulating a T cell-mediated immune response to atarget cell population or tissue in a mammal comprising the step ofadministering to the mammal a T cell that expresses a CAR, wherein theCAR comprises a binding moiety that specifically interacts with apredetermined target, a zeta chain portion comprising for example theintracellular domain of human CD3zeta, and a costimulatory signalingregion.

In one embodiment, the present invention includes a type of cellulartherapy where T cells are genetically modified to express a CAR and theCAR T cell is infused to a recipient in need thereof. The infused cellis able to kill tumor cells in the recipient. Unlike antibody therapies,CAR T cells are able to replicate in vivo resulting in long-termpersistence that can lead to sustained tumor control.

In one embodiment, the CAR T cells of the invention can undergo robustin vivo T cell expansion and can persist for an extended amount of time.In another embodiment, the CAR T cells of the invention evolve intospecific memory T cells that can be reactivated to inhibit anyadditional tumor formation or growth. For example, it was unexpectedthat the CART19 cells of the invention can undergo robust in vivo T cellexpansion and persist at high levels for an extended amount of time inblood and bone marrow and form specific memory T cells. Without wishingto be bound by any particular theory, CAR T cells may differentiate invivo into a central memory-like state upon encounter and subsequentelimination of target cells expressing the surrogate antigen.

Without wishing to be bound by any particular theory, the anti-tumorimmunity response elicited by the CAR-modified T cells may be an activeor a passive immune response. In addition, the CAR mediated immuneresponse may be part of an adoptive immunotherapy approach in whichCAR-modified T cells induce an immune response specific to the antigenbinding moiety in the CAR. For example, a CART19 cells elicits an immuneresponse specific against cells expressing CD19.

While the data disclosed herein specifically disclose lentiviral vectorcomprising anti-CD19 scFv derived from FMC63 murine monoclonal antibody,human CD8α hinge and transmembrane domain, and human 4-1BB and CD3zetasignaling domains, the invention should be construed to include anynumber of variations for each of the components of the construct asdescribed elsewhere herein. That is, the invention includes the use ofany antigen binding moiety in the CAR to generate a CAR-mediated T-cellresponse specific to the antigen binding moiety. For example, theantigen binding moiety in the CAR of the invention can target a tumorantigen for the purposes of treat cancer.

Cancers that may be treated include tumors that are not vascularized, ornot yet substantially vascularized, as well as vascularized tumors. Thecancers may comprise non-solid tumors (such as hematological tumors, forexample, leukemias and lymphomas) or may comprise solid tumors. Types ofcancers to be treated with the CARs of the invention include, but arenot limited to, carcinoma, blastoma, and sarcoma, and certain leukemiaor lymphoid malignancies, benign and malignant tumors, and malignanciese.g., sarcomas, carcinomas, and melanomas. Adult tumors/cancers andpediatric tumors/cancers are also included.

Hematologic cancers are cancers of the blood or bone marrow. Examples ofhematological (or hematogenous) cancers include leukemias, includingacute leukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavychain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia.

Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumors, such as sarcomas and carcinomas, include fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and othersarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreaticcancer, breast cancer, lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma,papillary thyroid carcinoma, pheochromocytomas sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas, medullarycarcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bileduct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer,testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors(such as a glioma (such as brainstem glioma and mixed gliomas),glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNSlymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brainmetastases).

In one embodiment, the antigen bind moiety portion of the CAR of theinvention is designed to treat a particular cancer. For example, the CARdesigned to target CD19 can be used to treat cancers and disordersincluding but are not limited to pre-B ALL (pediatric indication), adultALL, mantle cell lymphoma, diffuse large B-cell lymphoma, salvage postallogenic bone marrow transplantation, and the like.

In another embodiment, the CAR can be designed to target CD22 to treatdiffuse large B-cell lymphoma.

In one embodiment, cancers and disorders include but are not limited topre-B ALL (pediatric indication), adult ALL, mantle cell lymphoma,diffuse large B-cell lymphoma, salvage post allogenic bone marrowtransplantation, and the like can be treated using a combination of CARsthat target CD19, CD20, CD22, and ROR1.

In one embodiment, the CAR can be designed to target mesothelin to treatmesothelioma, pancreatic cancer, ovarian cancer, and the like.

In one embodiment, the CAR can be designed to target CD33/IL3Ra to treatacute myelogenous leukemia and the like.

In one embodiment, the CAR can be designed to target c-Met to treattriple negative breast cancer, non-small cell lung cancer, and the like.

In one embodiment, the CAR can be designed to target PSMA to treatprostate cancer and the like.

In one embodiment, the CAR can be designed to target Glycolipid F77 totreat prostate cancer and the like.

In one embodiment, the CAR can be designed to target EGFRvIII to treatgliobastoma and the like.

In one embodiment, the CAR can be designed to target GD-2 to treatneuroblastoma, melanoma, and the like.

In one embodiment, the CAR can be designed to target NY-ESO-1 TCR totreat myeloma, sarcoma, melanoma, and the like.

In one embodiment, the CAR can be designed to target MAGE A3 TCR totreat myeloma, sarcoma, melanoma, and the like.

However, the invention should not be construed to be limited to solelyto the antigen targets and diseases disclosed herein. Rather, theinvention should be construed to include any antigenic target that isassociated with a disease where a CAR can be used to treat the disease.

The CAR-modified T cells of the invention may also serve as a type ofvaccine for ex vivo immunization and/or in vive therapy in a mammal.Preferably, the mammal is a human.

With respect to ex vivo immunization, at least one of the followingoccurs in vitro prior to administering the cell into a mammal: i)expansion of the cells, ii) introducing a nucleic acid encoding a CAR tothe cells, and/or iii) cryopreservation of the cells.

Ex vive procedures are well known in the art and are discussed morefully below. Briefly, cells are isolated from a mammal (preferably ahuman) and genetically modified (i.e., transduced or transfected invitro) with a vector expressing a CAR disclosed herein. The CAR-modifiedcell can be administered to a mammalian recipient to provide atherapeutic benefit. The mammalian recipient may be a human and theCAR-modified cell can be autologous with respect to the recipient.Alternatively, the cells can be allogeneic, syngeneic or xenogeneic withrespect to the recipient.

The procedure for ex vivo expansion of hematopoietic stem and progenitorcells is described in U.S. Pat. No. 5,199,942, incorporated herein byreference, can be applied to the cells of the present invention. Othersuitable methods are known in the art, therefore the present inventionis not limited to any particular method of ex vivo expansion of thecells. Briefly, ex vivo culture and expansion of T cells comprises: (1)collecting CD34+ hematopoietic stem and progenitor cells from a mammalfrom peripheral blood harvest or bone marrow explants; and (2) expandingsuch cells ex vivo. In addition to the cellular growth factors describedin U.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3 andc-kit ligand, can be used for culturing and expansion of the cells.

In addition to using a cell-based vaccine in terms of ex vivoimmunization, the present invention also provides compositions andmethods for in vivo immunization to elicit an immune response directedagainst an antigen in a patient.

Generally, the cells activated and expanded as described herein may beutilized in the treatment and prevention of diseases that arise inindividuals who are immunocompromised. In particular, the CAR-modified Tcells of the invention are used in the treatment of CCL. In certainembodiments, the cells of the invention are used in the treatment ofpatients at risk for developing CCL. Thus, the present inventionprovides methods for the treatment or prevention of CCL comprisingadministering to a subject in need thereof, a therapeutically effectiveamount of the CAR-modified T cells of the invention.

The CAR-modified T cells of the present invention may be administeredeither alone, or as a pharmaceutical composition in combination withdiluents and/or with other components such as IL-2 or other cytokines orcell populations. Briefly, pharmaceutical compositions of the presentinvention may comprise a target cell population as described herein, incombination with one or more pharmaceutically or physiologicallyacceptable carriers, diluents or excipients. Such compositions maycomprise buffers such as neutral buffered saline, phosphate bufferedsaline and the like; carbohydrates such as glucose, mannose, sucrose ordextrans, mannitol; proteins; polypeptides or amino acids such asglycine; antioxidants; chelating agents such as EDTA or glutathione;adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions ofthe present invention are preferably formulated for intravenousadministration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

When “an immunologically effective amount”, “an anti-tumor effectiveamount”, “an tumor-inhibiting effective amount”, or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the T cells described herein may be administered at a dosageof 10⁴ to 10⁹ cells/kg body weight, preferably 10⁵ to 10⁶ cells/kg bodyweight, including all integer values within those ranges. T cellcompositions may also be administered multiple times at these dosages.The cells can be administered by using infusion techniques that arecommonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng.J. of Med. 319:1676, 1988). The optimal dosage and treatment regime fora particular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly.

In certain embodiments, it may be desired to administer activated Tcells to a subject and then subsequently redraw blood (or have anapheresis performed), activate T cells therefrom according to thepresent invention, and reinfuse the patient with these activated andexpanded T cells. This process can be carried out multiple times everyfew weeks. In certain embodiments, T cells can be activated from blooddraws of from 10 cc to 400 cc. In certain embodiments, T cells areactivated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc,80 cc, 90 cc, or 100 cc. Not to be bound by theory, using this multipleblood draw/multiple reinfusion protocol may serve to select out certainpopulations of T cells.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patientsubcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, by intravenous (i.v.) injection, orintraperitoneally. In one embodiment, the T cell compositions of thepresent invention are administered to a patient by intradermal orsubcutaneous injection. In another embodiment, the T cell compositionsof the present invention are preferably administered by i.v. injection.The compositions of T cells may be injected directly into a tumor, lymphnode, or site of infection.

In certain embodiments of the present invention, cells activated andexpanded using the methods described herein, or other methods known inthe art where T cells are expanded to therapeutic levels, areadministered to a patient in conjunction with (e.g., before,simultaneously or following) any number of relevant treatmentmodalities, including but not limited to treatment with agents such asantiviral therapy, cidofovir and interleukin-2, Cytarabine (also knownas ARA-C) or natalizumab treatment for MS patients or efalizumabtreatment for psoriasis patients or other treatments for PML patients.In further embodiments, the T cells of the invention may be used incombination with chemotherapy, radiation, immunosuppressive agents, suchas cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506,antibodies, or other immunoablative agents such as CAM PATH, anti-CD3antibodies or other antibody therapies, cytoxin, fludaribine,cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228,cytokines, and irradiation. These drugs inhibit either the calciumdependent phosphatase calcineurin (cyclosporine and FK506) or inhibitthe p70S6 kinase that is important for growth factor induced signaling(rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun.73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993). Ina further embodiment, the cell compositions of the present invention areadministered to a patient in conjunction with (e.g., before,simultaneously or following) bone marrow transplantation. T cellablative therapy using either chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, or antibodiessuch as OKT3 or CAMPATH. In another embodiment, the cell compositions ofthe present invention are administered following B-cell ablative therapysuch as agents that react with CD20, e.g., Rituxan. For example, in oneembodiment, subjects may undergo standard treatment with high dosechemotherapy followed by peripheral blood stem cell transplantation. Incertain embodiments, following the transplant, subjects receive aninfusion of the expanded immune cells of the present invention. In anadditional embodiment, expanded cells are administered before orfollowing surgery.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices. Thedose for CAMPATH, for example, will generally be in the range 1 to about100 mg for an adult patient, usually administered daily for a periodbetween 1 and 30 days. The preferred daily dose is 1 to 10 mg per dayalthough in some instances larger doses of up to 40 mg per day may beused (described in U.S. Pat. No. 6,120,766).

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

Example 1 T Cells Expressing Chimeric Receptors Establish Memory andPotent Antitumor Effects in Patients with Advanced Leukemia

Lymphocytes engineered to express chimeric antigen receptors (CARs) havedemonstrated minimal in vivo expansion and antitumor effects in previousclinical trials. The results presented herein demonstrate that that CART cells containing CD137 have potent non-cross resistant clinicalactivity following infusion in three of three patients treated withadvanced chronic lymphocytic leukemia (CLL). The engineered T cellsexpanded more than a thousand-fold in vivo, trafficked to bone marrowand continued to express functional CARs at high levels for at least 6months. On average, each infused CAR+ T cell eradicated at least 1000CLL cells. A CD19 specific immune response was demonstrated in the bloodand bone marrow, accompanied by complete remission in two of threepatients. A portion of the cells persist as memory CAR+ T cells,indicating the potential of this non-MHC restricted approach for theeffective treatment of B cell malignancies.

The materials and methods employed in these experiments are nowdescribed.

Materials and Methods

General Laboratory Statement

Research sample processing, freezing, and laboratory analyses wereperformed in the Translational and Correlative Studies Laboratory at theUniversity of Pennsylvania which operates under principles of GoodLaboratory Practice with established SOP and/or protocols for samplereceipt, processing, freezing, and analysis. Assay performance and datareporting conforms with MIATA guidelines (Janetzki et al., 2009,Immunity 31:527-528).

Protocol Design

The clinical trial (NCT01029366) was conducted as diagramed in FIG. 1.Patients with CD19 positive hematologic malignancy with persistentdisease following at least two prior treatment regimens and who were noteligible for allogeneic stem cell transplantation were eligible for thetrial. Following tumor restaging, peripheral blood T cells for CART19manufacturing were collected by apheresis and the subjects given asingle course of chemotherapy as specified in FIG. 10 during the weekbefore infusion. CART19 cells were administered by intravenous infusionusing a 3 day split dose regimen (10%, 30% and 60%) at the doseindicated in FIG. 10 and if available, a second dose was administered onday 10; only patient UPN 02 had sufficient cells for a second infusion.Subjects were assessed for toxicity and response at frequent intervalsfor at least 6 months. The protocol was approved by the US Food and DrugAdministration, the Recombinant DNA Advisory Committee and theInstitutional Review Board of the University of Pennsylvania. The firstday of infusion was set as study Day 0.

Subjects: Clinical Summary

The clinical summaries are outlined in FIG. 10 and detailed historiesare provided elsewhere herein. Patient UPN 01 was first diagnosed withstage II B cell CLL at age 55. The patient was asymptomatic and observedfor approximately 1½ years until requiring therapy for progressivelymphocytosis, thrombocytopenia, adenopathy, and splenomegaly. Over thecourse of time, the patient received prior lines of therapy. The mostrecent therapy was 2 cycles of pentostatin, cyclophosphamide andrituximab 2 months prior to CART19 cell infusion with a minimalresponse. The patient then received one cycle of bendamustine aslymphodepleting chemotherapy prior to CART-19 cell infusion.

Patient UPN 02 was first diagnosed with CLL at age 68 when the patientwas presented with fatigue and leukocytosis. The patient was relativelystable for 4 years when the patient developed progressive leukocytosis(195,000/μl), anemia and thrombocytopenia requiring therapy. Karyotypicanalysis showed that the CLL cells had deletion of chromosome 17p.Because of progressive disease, the patient was treated with alemtuzumabwith a partial response but within one and a half years the patient hadprogressive disease. The patient was retreated with alemtuzumab for 18weeks with a partial response and a 1 year progression free interval.The patient then received 2 cycles of bendamustine with rituximabwithout a significant response (FIG. 5A). The patient received singleagent bendamustine as lymphodepleting chemotherapy prior to CART-19 cellinfusion.

Patient UPN 03 presented at age 50 with asymptomatic stage I CLL and wasfollowed with observation for years. The patient had progressiveleukocytosis (white blood count 92,000/μl) and progressive adenopathyrequiring therapy. The patient received 2 cycles of rituximab withfludarabine that resulted in normalization of blood counts andsignificant improvement though not complete resolution in adenopathy.The patient had an approximately 3 year progression free interval.Karyotypic testing showed cells to contain deletion of chromosome 17pwith FISH demonstrating a TP53 deletion in 170 of 200 cells. Over thenext years the patient required 3 different lines of therapy (FIG. 10)for progressive leukocytosis and adenopathy, last receiving alemtuzumabwith a partial response 6 months prior CART19 cell infusion. The patientreceived pentostatin and cyclophosphamide as lymphodepletingchemotherapy prior to CART-19 cell infusion.

Vector Production

The CD19-BB-z transgene (GeMCRIS 0607-793) was designed and constructedas described (Milone et al., 2009, Mol Ther. 17:1453-1464). Lentiviralvector was produced according to current good manufacturing practicesusing a three-plasmid production approach at Lentigen Corporation asdescribed (Zufferey et al., 1997, Nature biotechnol 15:871-875).

Preparation of CART19 Cell Product

Methods of T cell preparation using paramagnetic polystyrene beadscoated with anti-CD3 and anti-CD28 monoclonal antibodies have beendescribed (Laport et al., 2003, Blood 102: 2004-2013). Lentiviraltransduction was performed as described (Levine et al., 2006, Proc NatlAcad Sci USA 103:17372-17377).

Methods for Tumor Burden Calculation

CLL burden at baseline was estimated as shown in FIG. 10. The amount ofCLL cells were calculated in bone marrow, blood, and secondary lymphoidtissues as described below.

Bone Marrow:

In healthy adults, the bone marrow represents approximately 5% of totalbody weight (Woodard et al., 1960, Phys Med Biol, 5:57-59; Bigler etal., 1976, Health Phys 31:213-218). The bone marrow in iliac crestsamples has an increasing percentage of inactive (fatty) marrow withage, rising from 20% of the total marrow at age 5 to about 50% by age35, when it remains stable until age 65, and then rises to about 67%inactive marrow by age 75 (Hartsock et al., 1965, Am J Clin Path43:326-331). The international reference value for the total skeletalweight of active (red) and inactive (fatty) marrow for males at age 35is currently set at 1170 g and 2480 g, respectively (Basic anatomicaland physiological data for use in radiological protection: The Skeletonin Annals of the ICRP, Vol. 25 (ed. Smith, H.) 58-68 (A report of a TaskGroup of Committee 2 of the International Commission on RadiologicalProtection, Oxford, 1995)). Adult males between ages 35 to 65 havemarrow that represents 5.0% total of body weight, comprised of 1.6% asactive (red) marrow and 3.4% as inactive (fatty) marrow (Basicanatomical and physiological data for use in radiological protection:The Skeleton in Annals of the ICRP. Vol. 25 (ed. Smith, H.) 58-68 (Areport of a Task Group of Committee 2 of the International Commission onRadiological Protection, Oxford, 1995)). Based on the bone marrow biopsyand aspirate specimens, the weight of CLL cells for the three patientsat baseline was calculated as shown in the Table 1. These estimates oftotal CLL marrow mass were then converted to total CLL cell number inthe marrow using 1 Kg=10¹² cells, and the resulting numbers are shown inFIG. 10. These calculations are based on the assumption that the CLL hasa uniform distribution in the bone marrow. For patient UPN 01,calculations are shown for a marrow biopsy that was obtained beforebendamustine chemotherapy, and for an aspirate obtained afterbendamustine and pre-CART19 infusion. The numbers are less precise forthe day-1 aspirate compared to the day −14 biopsy specimen due totechnical limitations of the day-1 aspirate. Patient UPN 02 had a singlepre-treatment biopsy specimen showing complete replacement of marrow byCLL. This patient had an unchanged specimen on day 30 post CART19. Themarrow burden for patient UPN 03 was calculated based on apost-chemotherapy and pre-CART19 biopsy.

TABLE 1 Marrow Mass WT of Inactive Total Wt of Active Marrow Marrowmarrow (kg) (kg) (kg) Normal males (ICRP 1.17 2.48 3.65 referencestandard) UPN 01 day −14 3.47 0.18 3.65 (95% cellular) UPN 02 day −473.47 0.18 3.65 (95% cellular) UPN 03 day −1 2.19 1.46 3.65 (60%cellular) Wt of CLL (kg) UPN 01 day −14 2.43 (70% CLL) UPN 01 day −11.73 (50% CLL by clot) UPN 02 day −47 3.29 (>95% CLL) UPN 03 day −1 0.88(40% CLL)

Blood:

Only patient UPN 02 had substantial CLL tumor burden in the bloodpre-CART19 infusion. Flow cytometry showed that the cells had a typicalphenotype as a clonal population with a dim surface kappa-restrictedCD5+CD10-CD19+CD20(dim)+CD23(variable)+IgM-B cell population.Approximately 35% of the CLL cells coexpressed CD38. The CLL burden didnot clear with 3 cycles of bendamustine chemotherapy and was present atthe time of CART19 infusions. At the time of CART19 infusion, the CLLcount in blood was 55,000 cells/μL. Assuming a blood volume of 5.0 L,patient UPN 02 had 2.75×10¹¹ CLL cells in blood on day 0. Given thenormal overall WBC in patients UPN 01 and 03, the circulating diseaseburden in these patients was not calculated, which would lead to aslight underestimate of total body burden.

Secondary Lymphoid Tissues:

The volume of lymphadenopathy and splenomegaly was quantified on axialCT scans using FDA-approved software. The volumes are for chest, abdomenand pelvis only. Masses from the T1 vertebral body to the level of thebifurcation of the common femoral artery were measured in all patients,and in some, the nodes in the inguinal area were also included. Nodes inthe head/neck and extremities were excluded from analysis and excludedfrom the baseline CLL target cell number, which would also lead to aslight underestimate of total body burden. Patients UPN 01 and 03 havehad sustained complete remissions beyond 6 months, and thus the formula(baseline volume-month 3 volume) was used to determine the reduction intumor burden from baseline: patient UPN 02 had stable disease inadenopathy, and thus the baseline tumor mass is estimated by subtractingthe reference splenic volume from age matched healthy males (Harris etal., 2010, Eur J Radiol 75:e97-e101). Baseline tumor mass was convertedto CLL cells using a density approach (1 Kg/L density, and 1Kg=1012cells) cells or a volume approach (CLL cells are 10 μM diameteror 600 fL, assuming spherical shape), and both values presented in FIG.10. The tumor volumes in secondary lymphoid tissues in the threepatients are shown below in Table 2 as calculated from the available CTscans.

TABLE 2 Tumor Volumes LN volume Spleen volume Total volume Patient StudyDay (mm3) (mm3) (mm3) UPN −37 239655 1619180 1858835 01 1 month 1050051258575 1363580 3 month 65060 1176625 1241685 UPN −24 115990 11668001282790 02 1 month 111755 940960 1052715 UPN −10 239160 435825 674985 031 month 111525 371200 482725 3 month 47245 299860 347105

The baseline CT scan for patient UPN 01 was performed 8 days after 2cycles of pentostatin/cyclophosphamide/rituximab, and showed no responseto this chemotherapy regimen compared to the previous CT scan. Thepatient had one cycle of bendamustine before CART19, and thus, thechange in tumor volume from Day −37 to Day+31 for UPN 01 cannot excludethe potential contribution of the bendamustine as well as CART19.Similarly, the change in tumor volume for UPN 03 reflects the combinedeffect of 1 cycle of pentastatin/cyclophosphamide and CART19.

Method for Estimating Effective In Vivo E:T Ratio in Patients

The E:T ratio of infused CAR T cells to the number of tumor cells killedwas calculated using the number of tumor cells present at the time ofCAR T cell injection and the number of CAR T cells injected (Carpenitoet al., 2009, Proc Natl Acad Sci USA 106:3360-3365). For the presentinvention, the number of CART19+ T cells injected as shown on FIG. 10was used because it is not possible to determine the absolute number ofCART19+ T cells present in vivo with sufficient accuracy or precision.The available data on CART19 expansion in blood and marrow is robust asdepicted in FIG. 2 and FIG. 6. However it was not possible to determinethe trafficking of CART19 to other sites such as secondary lymphoidtissues, creating substantial uncertainty on the total number of CART19cells achieved in vivo at the time of maximal tumor reduction. Thecalculated values from Table 3 were used to derive the effective E:Tratios.

TABLE 3 Calculated CART19 E:T ratios achieved in vivo Tumor Burden(Baseline and Delta) Bone Nodes/ Total Change CART19+ marrow BloodSpleen¹ in CLL cells In Vivo Patient Baseline Baseline Baseline BurdenInfused E:T UPN 01 1.70E+12 N/A 8.1E+11 2.51E+12 1.13E+09 1:2200 UPN 023.20E+12 2.75E+11 1.6E+12  2.74E+11² 5.80E+08 1:1000 UPN 03 8.80E+11 N/A4.4E+11 1.32E+12 1.42E+07  1:93,000 Range 1000-93,000 ¹average ofdensity and volume method ²Patient UPN02 did not respond in bone marrowand had a partial reduction in adenopathy (3.1E+11 cells) in the tumormasses measured by CT in spleen and lymph nodes. See FIG. 5A forresponse in blood.

Sample Processing and Freezing

Samples (peripheral blood, marrow) were collected in lavender top(K2EDTA,) or red top (no additive) vacutainer tubes (Becton Dickinson)and delivered to the TCSL within 2 hours of draw. Samples were processedwithin 30 minutes of receipt according to established laboratory SOP.Peripheral blood and marrow mononuclear cells were purified via Ficolldensity gradient centrifugation using Ficoll-Paque (GE Health care,17-1440-03) and frozen in RPMI (Gibco 11875-135) supplemented with 4%human serum albumin (Gemini Bio-Products, 800-120), 2% Hetastarch(Novaplus, NDCO409-7248-49), and 10% DMSO (Sigma, D2650) using 5100 Cryo1° freezing containers: after 24-72 hours at −80° C., cells weretransferred to liquid Nitrogen for long-term storage. Apheresis sampleswere obtained through the Hospital of the University of PennsylvaniaBlood Bank and processed in the CVPF by Ficoll gradient purification andfrozen as above. Viability immediately post-thaw was greater than 85%when assessed. For serum isolation, samples were allowed to coagulatefor 1.5-2 hours at room temperature; serum isolated by centrifugation,and single use 100 μl aliquots frozen at −800° C.

Cell Lines

K562 (CML, CD19-negative) was obtained from ATCC (CCL-243). K562/CD19, agenerous gift of Carmine Carpenito, and is K562 lentivirally transducedat 100% frequency to express the CD19 molecule. NALM-6, a CD19-positivenon-T, non-B ALL precursor B cell line (Hurwitz et al., 1979, Int JCancer 23:174-180), and confirmed to express the CD19 antigen was agenerous gift of Laurence Cooper. The above cell lines were maintainedin RIO medium (RPMI 1640 (Gibco, 11875) supplemented with 10% fetalbovine serum (Hyclone), and 1% Pen-Strep (Gibco, 15140-122). Peripheralmononuclear cells (ND365) from a healthy donor were obtained byapheresis from the Human Immunology Core at the University ofPennsylvania, processed, and frozen as above.

DNA Isolation and Q-PCR Analysis

Whole-blood or marrow samples were collected in lavender top (K3EDTA) BDvacutainer tubes (Becton Dickinson). Genomic DNA was isolated directlyfrom whole-blood using QIAamp DNA blood midi kits (Qiagen) andestablished laboratory SOP, quantified by spectrophotometer, and storedat −80° C. Q-PCR analysis on genomic DNA samples was performed in bulkusing 123-200 ng genomic DNA/time-point. ABI Taqman technology and avalidated assay to detect the integrated CD19 CAR transgene sequence.Pass/fail parameter ranges, including standard curve slope and r²values, ability to accurately quantify a reference sample (1000copies/plasmid spike) and no amplification in healthy donor DNA samplewere calculated from the qualification studies and pre-establishedacceptance ranges. Primer/probes for the CD19 CAR transgene were asdescribed (Milone et al., 2009, Mol Ther 17:1453-1464). To determinecopy number/unit DNA an 8-point standard curve was generated consistingof 10⁶-5 copies lentivirus plasmid spiked into 100 ng non-transducedcontrol genomic DNA. Each data-point (samples, standard curve, referencesamples) was evaluated in triplicate with average values reported. Forpatient UPN 01, all reported values were derived from a positive Ctvalue in 3/3 replicates with % CV less than 0.46%. For patient UPN 02,with the exception of the day +177 sample (⅔ replicates positive, high %CV), all reported values were derived from a positive Ct value in 3/3replicates with % CV less than 0.72%. For patient UPN 03, with theexception of the day +1 sample (⅔ replicates positive, 0.8% CV) and theday +3 sample (⅔ replicates positive, 0.67% CV), all reported valueswere derived from a positive Ct value in 3/3 replicates with % CV lessthan 1.56%. The lower limit of quantification (LLOQ) for the assay wasdetermined from the standard curve at 2 copies/microgram DNA (10copies/200 ng input DNA); average values below LLOQ (i.e. reportable notquantifiable) are considered approximate. A parallel amplificationreaction to control for the quality of interrogated DNA was performedusing 12-20 ng input genomic DNA, a primer/probe combination specificfor non-transcribed genomic sequence upstream of the CDKN1A gene(GENEBANK: Z85996) (sense primer: GAAAGCTGACTGCCCCTATTTG; SEQ ID NO. 25,antisense primer: GAGAGGAAGTGCTGGGAACAAT: SEQ ID NO. 26, probe: VIC-CTCCCC AGT CTC TTT: SEQ ID NO. 27), and an 8 point standard curve createdby dilution of control genomic DNA: these amplification reactionsproduced a correction factor (CF) (ng detected/ng input). Copiestransgene/microgram DNA were calculated according to the formula: copiescalculated from CD19 standard curve/input DNA (ng)×CF×1000 ng. Accuracyof this assay was determined by the ability to quantify marking of theinfused cell product by Q-PCR according to the formula: Averagemarking=detected copies/input DNA×6.3 pg DNA/male somatic cell×CF versustransgene positivity by flow cytometry using CAR-specific detectionreagents. These blinded determinations generated 22.68% marking for theUPN 01 infusion product (22.6% by flow cytometry), 32.33% marking forUPN 02 infusion product (23% by flow cytometry), and 4.3% marking forthe UPN 03 infusion product (4.7% marking by flow cytometry).

Cytokine Analyses

Quantification of soluble cytokine factors was performed using Luminexbead array technology and kits purchased from Life technologies(Invitrogen). Assays were performed as per the manufacturer protocolwith an 8 point standard curve generated using a 3-fold dilution series.Each standard point and sample was evaluated in duplicate at 1:3dilution: calculated % CV for the duplicate measures were less than 15%.Data were acquired on a Bioplex 200 and analyzed with Bioplex Managerversion 5.0 software using 5-parameter logistic regression analysis.Standard curve quantification ranges were determined by the 80-120%(observed/expected value) range. Individual analyte quantificationranges are reported in the Figure legends.

Cellular assay to detect CAR function Cells were evaluated forfunctionality after thaw and overnight rest in TCM by measuring CD107degranulation in response to target cells. Degranulation assays wereperformed using 1×10⁶PBMC and 0.25×10⁶ target cells in a final volume of500 μl in 48-well plates for 2 hours at 37° C. in the presence of CD49d(Becton Dickinson), anti-CD28, monensin (e-Bioscience) and CD107a-FITCantibody (eBiosciences) essentially as described (Betts et al., 2003, JImmunol Methods 281:6578).

Antibody Reagents

The following antibodies were used for these studies: MDA-CAR, a murineanti CD19 CAR antibody conjugated to Alexa647 was a generous gift ofDrs. Bipulendu Jena and Laurence Cooper (MD Anderson Cancer Center). Formulti-parametric immunophenotyping and functional assays: anti-CD3-A700,anti-CD8-PE-Cy7, anti-PD-1-FITC anti-CD25-AF488, anti-CD28-PercP-Cy5.5,anti-CD57-eF450, anti-CD27-APC-eF780, anti-CD17-APC-eF780,anti-CD45RA-eF605NC, CD107a-FITC (all from e-Bioscience),anti-CD4-PE-Texas Red and Live/Dead Aqua (from Life Technologies) andanti-CD14-V500, anti-CD16-V500 (from Becton Dickinson). For generalimmunophenotyping: CD3-PE, CD14-APC, CD14-PE-Cy7, CD16-FITC, CD16PE-Cy7,CD19-PE-Cy7, CD20-PE, all from Becton Dickinson.

Multi-Parameter Flow Cytometry

Cells were evaluated by flow cytometry either fresh after Ficoll-Paqueprocessing or, if frozen, after overnight rest at a density of2×10⁶cells/ml in T cell medium (TCM) (X-vivo 15 (Lonza, 04-418Q)supplemented with 5% human AB serum (GemCall, 100-512), 1% Hepes (Gibco,15630-080), 1% Pen-Strep (Gibco, 15140-122), 1% Glutamax (Gibco,35050-061), and 0.2% N-Acetyl Cysteine (American Regent,NDCO517-7610-03). Multi-parametric immunophenotyping was performed on4×10⁶ total cells/condition, using FMO stains as described in the text.Cells were stained at a density of 1×10⁶ cells/100 μl PBS for 30 minuteson ice using antibody and reagent concentrations recommended by themanufacturer, washed, re-suspended in 0.5% paraformaldehyde and acquiredusing a modified LSRII (BD Immunocytometry systems) equipped with Blue(488 nm) Violet (405 nm), Green (532), and Red (633 nm) lasers andappropriate filter sets for the detection and separation of the aboveantibody combinations. A minimum of 100,000 CD3+ cells were acquired)for each stain. For functional assays, cells were washed, stained forsurface markers, re-suspended in 0.5% paraformaldehyde and acquired asabove; a minimum of 50,000 CD3+ events were collected for each stainingcondition. Compensation values were established using single antibodystains and BD compensation beads (Becton Dickinson) and were calculatedand applied automatically by the instrument. Data were analyzed usingFlowJo software (Version 8.8.4, Treestar). For general immunophenotypingcells were acquired using an Accuri C6 cytometer equipped with a Blue(488) and Red (633 nm) laser. Compensation values were established usingsingle antibody stains and BD compensation beads (Becton Dickinson) andwere calculated manually. Data were analyzed using C-Flow softwareanalysis package (version 1.0.264.9. Accuri cytometers).

Patient Past Medical Histories and Response to Therapy

The clinical treatment summaries are outlined in FIG. 10. Patient UPN 01was first diagnosed with stage II B cell CLL at age 55. The patient wasasymptomatic and observed for approximately 1½ years until requiringtherapy for progressive lymphocytosis, thrombocytopenia, adenopathy, andsplenomegaly. After 4 cycles of fludarabine the patient had completenormalization of blood counts and a complete response by CT scans.Progression was noted within 5 months with asymptomatic lymphocytosis,thrombocytopenia, and increasing adenopathy. The patient was observedwithout symptoms for approximately 3 years, and later required treatmentwith Rituximab and fludarabine for progressive leukocytosis, anemia, andthrombocytopenia. The patient was treated with 4 cycles of rituximabwith fludarabine with partial improvement in blood counts. The patientagain had progression within one year requiring therapy manifested byleukocytosis (WBC 150,000/μl) and thrombocytopenia (platelets 30,000/μl)and was treated with alemtuzumab with normalization of blood counts.Progression was noted within 13 months. The patient then received singleagent rituximab without a significant response and followed byrituximab, cyclophosphamide, vincristine, and prednisone (R-CVP) for 2cycles with minimal response and followed by lenalidomide. Lenalidomidewas discontinued because of toxicity. The patient received 2 cycles ofpentostatin, cyclophosphamide and rituximab with a minimal response.

Later, the patient received bendamustine as lymphodepleting chemotherapy4 days prior to CART19 cell infusion. Prior to therapy, WBC was14,200/μl, hemoglobin 11.4 gm/dl, platelet count 78,000/μl and ALC was8000/μl. The CT scan showed diffuse adenopathy and bone marrow wasextensively infiltrated with CLL (67% of cells). The patient received1.6×10⁷ CART-19 cells/kg (1.13×10⁹ total CART19 cells as assessed byFACS). There were no infusional toxicities. The patient becameneutropenic approximately 10 days after bendamustine and 6 days afterCART19 cell infusions, and beginning 10 days after the first CART19infusion, the patient developed fevers, rigors and transienthypotension. At the same time, a chest X-ray and CT scan demonstrated aleft upper lobe pneumonia treated with antibiotics. The fevers persistedfor approximately 2 weeks and resolved when there was neutrophilrecovery. The patient has had no further infectious or constitutionalsymptoms.

The patient achieved a rapid and complete response as depicted in FIG.5. Between 1 and 6 months after infusion no circulating CLL cells havebeen detected in the blood by flow cytometry. Bone marrow at 1, 3 and 6months after CART-19 cell infusions shows sustained absence of thelymphocytic infiltrate by morphology and flow cytometry testing. The CTscans at 1 and 3 months after infusion show complete resolution ofabnormal adenopathy. The patient has had a persistent leukopenia (WBC1000-3900/ul) and thrombocytopenia (platelets ˜100,000/ul), and mildhypogammaglobulinia (IgG 525 mg/dL, normal 650-2000 mg/dL) but noinfectious complications.

Patient UPN 02 was treated with CART19 cells at age 77. The patient hada relevant history of coronary artery disease and was first diagnosedwith CLL in 2000 at age 68 when the patient presented with fatigue andleukocytosis. The patient was relatively stable for 4 years when thepatient developed progressive leukocytosis (195,000/μl), anemia andthrombocytopenia requiring therapy. Genetic testing at that time showedthat the CLL cells had deletion of chromosome 17p. Because ofprogressive disease, the patient was treated with a 12 week course ofalemtuzumab with a partial response and improvement in blood counts.Within one and a half years the patient had progressive leukocytosis,anemia, thrombocytopenia, and splenomegaly. Kalyotypic analysisconfirmed deletion of chromosome 17p now with a deletion of chromosome13q. The patient was retreated with alemtuzumab for 18 weeks withimprovement of leukocytosis and stabilization of anemia andsplenomegaly. The patient had evidence of progressive leukocytosis,anemia, and thrombocytopenia within one year. Treatment included 2cycles of bendamustine with rituximab resulting in stable disease but nosignificant improvement as shown in FIG. 5A.

The patient received bendamustine alone as lymphodepleting chemotherapyprior to CART-19 cell infusion. The patient received 4.3×10⁶ CART19cells/kg (4.1×10⁸ total cells) in 3 split infusions complicated bytransient fevers as high as 102° degrees for 24 hours. On day 11 afterthe first infusion, the patient received a boost of 4.1×10⁸ (4.3×10⁶/kg)CART19 cells and this infusion was complicated by fevers, rigors andshortness of breath without hypoxia requiring a 24 hour hospitalization.There was no evidence for cardiac ischemia, and the symptoms resolved.On day 15 after the first CART-19 infusion and day 4 after the boostCART19 cell infusion the patient was admitted to the hospital with highfevers (up to 104° F.), chills and rigors. Extensive testing with bloodand urine cultures and CXR failed to identify a source of infection. Thepatient complained of shortness of breath but had no hypoxia. Anechocardiogram showed severe hypokinesis. Ejection fraction was 20%. Thepatient received prednisone 1 mg per kilogram for one day and 0.3 mg perkilogram for approximately one week. This resulted in rapid resolutionof fevers and cardiac dysfunction.

Coincident with the onset of high fevers, the patient had a rapid dropin lymphocytes from peripheral blood as depicted in FIG. 5A. Althoughthe patient had normalization of white blood count, the patient hadpersistent circulating CLL, stable moderate anemia and thrombocytopenia.Bone marrow showed persistent extensive infiltration of CLL one monthafter therapy despite dramatic peripheral blood cytoreduction, and CTscans showed a partial reduction of adenopathy and splenomegaly. Fivemonths after CART19 cell infusions the patient developed progressivelymphocytosis. Nine months after infusions the patient has lymphocytosis(16,500/μl) with stable modest anemia and thrombocytopenia with stableadenopathy. The patient remains asymptomatic and has not had furthertherapy.

Patient UPN 03 was diagnosed with asymptomatic stage I CLL at age 50 andwas followed with observation for 6 years. Later, the patient hadprogressive leukocytosis (white blood count 92,000/μl) and progressiveadenopathy requiring therapy. The patient received 2 cycles of rituximabwith fludarabine that resulted in normalization of blood counts andsignificant improvement though not complete resolution in adenopathy.The patient had approximately a 3 year progression free intervalfollowed over the next 6 months by rapidly progressive leukocytosis (WBC165,000/μl) and progressive adenopathy requiring therapy. The patientreceived one cycle of fludarabine and 3 cycles of rituximab withfludarabine with normalization of blood counts and resolution ofpalpable adenopathy. The patient had an approximate 20 month progressionfree interval until the patient again developed rapidly progressingleukocytosis and adenopathy. At this time, bone marrow was extensivelyinfiltrated with CLL and karyotypic analysis showed cells to containdeletion of chromosome 17p with FISH demonstrating a TP53 deletion in170/200 cells. The patient received one cycle of rituximab withbendamustine followed by 4 cycles of bendamustine only (due to a severeallergic reaction to rituximab). The patient had initial normalizationof blood counts but shortly after discontinuation of therapy hadprogressive leukocytosis and adenopathy.

Autologous T cells were collected by apheresis and cryopreserved fromPatient UPN3. The patient was then treated with alemtuzumab for 11 weeksthrough with an excellent hematologic response. There was improvementthough not complete resolution in adenopathy. The patient had active butstable disease over the next 6 months. Later, the patient receivedpentostatin and cyclophosphamide as lymphodepleting chemotherapy priorto CART19 cell infusion.

Three days after chemotherapy but prior to cell infusion, the bonemarrow was hypercellular (6(%) with approximately 40% involvement byCLL. Because of manufacturing limitations inherent in apheresiscollections from CLL patients as depicted in Table 3 and (Bonyhadi etal., 2005, J Immunol 174:2366-2375), the patient was infused with atotal of 1.46×10CART19+ cells per kg (1.42×10⁷ total CART19+ cells) over3 days. There were no infusional toxicities. Fourteen days after thefirst infusion, the patient began having chills, fevers as high as 102°F. rigors, nausea and diarrhea treated symptomatically. The patient hadno respiratory or cardiac symptoms. By day 22 after infusion, a tumorlysis syndrome was diagnosed manifested by an elevated LDH, uric acid,and complicated by renal insufficiency. The patient was hospitalized andtreated with fluid resuscitation and rasburicase with rapidnormalization of uric acid and renal function. A detailed clinicalevaluation with a CXR, blood, urine, and stool cultures were performedand were all negative or normal.

Within 1 month of CART-19 infusions, the patient had clearance ofcirculating CLL from the blood and bone marrow by morphology, flowcytometry, cytogenetic, and FISH analysis and CT scans showed resolutionof abnormal adenopathy (FIG. 5C). The patient's remission has beensustained beyond 8 months from the initial CART19 cell infusion.

The results of the experiments are now described.

Clinical Protocol

Three patients with advanced, chemotherapy-resistant CLL were enrolledon a pilot clinical trial as depicted in FIG. 1. All patients wereextensively pretreated with various chemotherapy and biologic regimensas shown in FIG. 10. Two of the patients had p53 deficient CLL, adeletion that portends poor response to conventional therapy and rapidprogression (Dohner et al., 1995, Blood, 851580-1589). Each of thepatients had large tumor burdens following the preparative chemotherapy,including extensive marrow infiltration (40 to 95%) and lymphadenopathy;patient UPN 02 also had significant peripheral lymphocytosis. The CART19T cells were prepared as depicted in FIG. 1B and details of the cellmanufacturing and product characterization for each patient are shown inTable 4. All patients were pretreated 1-4 days before CART19 T cellinfusions with lymphodepleting chemotherapy. A split dose cell infusionschedule was used because the trial testing a CAR incorporating a 4-1BBcostimulatory signaling domain as depicted in FIG. 1A.

TABLE 4 Apheresis products and CART19 product release criteria AssaySpecification UPN 01 UPN 02 UPN 03 Apheresis Flow N/A 4.46% 2.29% 2.67%Product Cytometry For CD3+ of CD45+ CART19 Total Cell −2-5 × 10⁹ 5 × 10⁹1.275 × 10⁹ 3 × 10⁹ Product Number 1.275 × 10⁹ Infused [2.55 × 10⁹total] Cell Viability >=70% 96.2% 95.3 (90.5)¹ 90.3 % CD3+ Cells >=80%88.9% 98.8 98.9 Residual <=100 beads/ 3.95  1  4 Bead # 3 × 10⁶ CellsEndotoxin <=3.5 EU/mL <0.5 EU/mL <0.5 EU/mL <0.5 EU/mL MycoplasmaNegative Negative Negative Negative Sterility No Growth No Growth NoGrowth No Growth (Bactec) Fungal No Growth No Growth No Growth No GrowthCulture BSA ELISA <=1 μg/mL <0.5 ng/mL <0.5 ng/mL <0.5 ng/mL ReplicationRCL Not Inconclusive² Inconclusive² Competent Not Detectable DetectableLentivirus (RCL) Transduction >=20% 22.6% 23% 4.74%⁴ Efficiency (scFvExpression) Vector DNA 0.2-3 0.15³  0.275  0.101 Sequence copies/cell(CART19 PCR) ¹= Dose #2. ²= Assay value at Day 12 below LOQ and had beendecreasing from earlier in expansion consistent with carryover ofplasmid DNA from vector generation. Submitted to the FDA as aninformational amendment. ³= Product release based on surface staining byFACS. ⁴= Treatment exception granted for release criteria by externalDSMC and IRB.

In Vivo Expansion and Persistence of CART19 and Trafficking to BoneMarrow

CAR+ T cells expanded using CD3/CD28 beads and expressing a 4-1BBsignaling domain is believed to be in improvement to CARs lacking 4-1BB.A Q-PCR assay was developed to enable quantitative tracking of CART19cells in blood and bone marrow. All patients had expansion andpersistence of the CART19-cells in blood for at least 6 months asdepicted in FIGS. 2A and 2C. Notably, patients UPN 01 and UPN 03 had a1,000 to 10,000 fold expansion of CAR+ T cells in blood during the firstmonth post infusion. The peak expansion levels coincided with onset ofthe post-infusion clinical symptoms in patient UPN 01 (day 15) andpatient UPN 03 (day 23). Furthermore, following an initial decay thatcan be modeled with first order kinetics, the CART19 T cell levelsstabilized in all 3 patients from day 90 to 180 post infusion.Significantly, the CART19 T cells also trafficked to bone marrow in allpatients, albeit at 5- to 10-fold lower levels than observed in blood asdepicted in FIGS. 2D through 2F. Patients UPN 01 and 03 had a log lineardecay in the marrow, with a disappearance T½ of ˜35 days.

Induction of Specific Immune Responses in the Blood and Bone MarrowCompartments Following CART19 Infusion

Serum samples from all patients were collected and batch analyzed toquantitatively determine cytokine levels, assessing a panel ofcytokines, chemokines, and other soluble factors to assess potentialtoxicities and to provide evidence of CART19 cell function as depictedin FIG. 3. Of thirty analytes tested, eleven had a 3-fold or more changefrom baseline, including 4 cytokines (IL-6, INF-γ, IL-8 and IL-10), 5chemokines (MIP-1α, MIP-1β, MCP-1, CXCL9, CXCL10) and soluble receptorsfor IL-1Rα and IL-2Rα. Of these, interferon-γ had the largest relativechange from baseline. Interestingly, the peak time of cytokine elevationin UPN 01 and UPN 03 correlated temporally with the previously describedclinical symptoms and the peak levels of CART19 cells in the blood ineach patient. Only modest changes were noted in patient UPN 02, perhapsas a result of corticosteroid treatment given to this patient. Elevationof soluble IL-2 was not detected in the serum of the patients, eventhough one of the pre-clinical rationales for developing CAR+ T cellswith 4-1BB signaling domains was the reduced propensity to trigger IL-2secretion compared to CD28 signaling domains (Milone et al., 2009, MolTher. 17:1453-1464). This may be relevant to sustained clinical activityas previous studies have shown that CAR+ T cells are potentiallysuppressed by regulatory T cells (Lee et al., 2011, Cancer Res71:2871-2881), cells that could be elicited by CARs that secretesubstantial amounts of IL-2 or by the provision of exogenous IL-2post-infusion. Finally, a robust induction of cytokine secretion in thesupernatants from bone marrow aspirates of UPN 03 was observed asdepicted in FIG. 3D that also coincided with the development of tumorlysis syndrome and complete remission.

Prolonged Expression and Establishment of a Population of Memory CART19Cells in Blood

A central question in CAR-mediated cancer immunotherapy is whetheroptimized cell manufacturing and costimulation domains enhance thepersistence of genetically modified T cells and permit the establishmentof CAR+ memory T cells in patients. Previous studies have notdemonstrated robust expansion, prolonged persistence and/or expressionof CARs on T cells after infusion (Kershaw et al., 2006, Clin Cancer Res12:6106-6115; Lamers et al., 2006, J Clin Oncol 24:e20-e22; Till et al.,2008, Blood, 112, 2261-2271; Savoldo et al., 2011, J Clin Investdoi:10.1172/JCI46110). Flow-cytometric analysis of samples from bothblood and marrow at 169 days post infusion revealed the presence ofCAR19 expressing cells in UPN 03 (FIGS. 4A and 4B), and an absence of Bcells as depicted in FIG. 4A. Notably, by Q-PCR assay, all threepatients have persisting CAR+ cells at 4 months and beyond as depictedin FIG. 2 and FIG. 6. The in vivo frequency of CAR+ cells by flowcytometry closely matched the values obtained from the PCR assay for theCART19 transgene. Importantly, in patient UPN 03, only CD3+ cellsexpressed the CAR19, as CAR19+ cells were not detectable in CD16- orCD14-positive subsets as depicted in FIG. 4A. CAR expression was alsodetected on the surface of 4.2% of T cells in the blood of patient UPN01 on day 71 post infusion as depicted in FIG. 7.

Next, polychromatic flow cytometry was used to perform detailed studiesto further characterize the expression, phenotype, and function ofCART19 cells in UPN 03 using an anti-CAR idiotype antibody (MDA-647) anda gating strategy shown in FIG. 8. Notable differences in the expressionof memory and activation markers in both CD8+ and CD4+ cells based onCAR19 expression was observed. At day 56, CART19 CD8+ cells displayedprimarily an effector memory phenotype (CCR7-CD27-CD28-) consistent withprolonged and robust exposure to antigen as depicted in FIG. 4C. Incontrast, CAR-negative CD8+ cells consisted of mixtures of effector andcentral memory cells, with CCR7 expression in a subset of cells, andsubstantial numbers in the CD27+/CD28− and CD27+/CD28+ fractions. Whileboth CART19 and CAR-negative cell populations substantially expressedCD57, this molecule was uniformly co-expressed with PD-1 in the CART19cells, a possible reflection of the extensive replicative history ofthese cells. In contrast to the CAR-negative cell population, theentirety of the CART19 CD8+ population lacked expression of both CD25and CD127. By day 169, while the phenotype of the CAR-negative cellpopulation remained similar to the day 56 sample, the CART19 populationhad evolved to contain a minority population with features of centralmemory cells, notably expression of CCR7, higher levels of CD27 andCD28, as well as CAR+ cells that were PD-1-negative, CD57-negative andCD127-positive.

In the CD4+ compartment, at day 56 CART19 cells were characterized byuniform lack of CCR7 and a predominance of CD27+/CD28+/PD-1+ cellsdistributed within both CD57+ and -compartments, and an essentialabsence of CD25 and CD127 expression as depicted in FIG. 4B. Incontrast, CAR-negative cells at this time-point were heterogeneous inCCR7, CD27 and PD-1 expression, expressed CD127 and also contained asubstantial CD25+/CD127-(potential regulatory T cell) population. By day169, while CD28 expression remained uniformly positive in all CAR+CD4+cells, a fraction of the CART19 CD4+ cells had evolved toward a centralmemory phenotype with expression of CCR7, a higher percentage ofCD27-cells, the appearance of a PD-1-negative subset, and acquisition ofCD127 expression. CAR-negative cells remained reasonably consistent withtheir day 56 counterparts, with the exception of a reduction in CD27expression a decrease in the percentage of CD25+/CD127-cells.

CART19 Cells can Retain Effector Function after 6 Months in Blood

In addition to short persistence and inadequate in vivo proliferation, alimitation of previous trials with CAR+ T cells has been the rapid lossof functional activity of the infused T cells in vivo. The high levelCART19 cell persistence and surface expression of the CAR19 molecule inpatient UPN 01 and 03 provided the opportunity to directly testanti-CD19-specific effector functions in cells recovered fromcryopreserved peripheral blood samples. PBMC from patient UPN 03 werecultured with target cells that were either positive or negative forCD19 expression (FIG. 4d ). Robust CD19-specific effector function ofCART19 T cells was demonstrated by specific degranulation againstCD19-positive but not CD19-negative target cells, as assessed by surfaceCD107a expression. Notably, exposure of the CART19 population toCD19-positive targets induced a rapid internalization of surface CAR-19as depicted in FIG. 8 for surface expression of CAR19 in the sameeffector cells in standard flow-cytometric staining. The presence ofcostimulatory molecules on target cells was not required for triggeringCART19 cell degranulation because the NALM-6 line does not express CD80or CD86 (Brentjens et al., 2007, Clin Cancer Res 13:5426-5435). Effectorfunction was evident at day 56 post infusion and was retained at the day169 time-point. Robust effector function of CAR+ and CAR-T cells couldalso be demonstrated by pharmacologic stimulation.

Clinical Activity of CART19 Cells

There were no significant toxicities observed during the four daysfollowing the infusion in any patient, other than transient febrilereactions. However, all patients subsequently developed significantclinical and laboratory toxicities between day 7 and 21 following thefirst infusion. These toxicities were short-term and reversible. Of thethree patients treated to date, there are 2 CRs and 1 PR at >6 monthspost CART19 infusion according to standard criteria (Hallek et al.,2008, Blood 111:5446). Details of past medical history and response totherapy for each patient are depicted in FIG. 10.

In brief, patient UPN 01 developed a febrile syndrome, with rigors andtransient hypotension beginning 10 days after infusion. The feverspersisted for approximately 2 weeks and resolved: the patient has had nofurther constitutional symptoms. The patient achieved a rapid andcomplete response as depicted in FIG. 5. Between 1 and 6 months afterinfusion, no circulating CLL cells have been detected in the blood byflow cytometry. Bone marrow at 1, 3, and 6 months after CART19 cellinfusions shows sustained absence of the lymphocytic infiltrate bymorphology and flow cytometric analysis as depicted in FIG. 5B. CT scansat 1 and 3 months after infusion show resolution of adenopathy asdepicted in FIG. 5C. Complete remission was sustained for 10+ months atthe time of this report.

Patient UPN 02 was treated with 2 cycles of bendamustine with rituximabresulting in stable disease as depicted in FIG. 5A. The patient receiveda third dose of bendamustine as lymphodepleting chemotherapy prior toCART19 T cell infusion. The patient developed fevers to 40° C., rigorsand dyspnea requiring a 24 hour hospitalization on day 11 after thefirst infusion and on the day of the second CART19 cell boost. Feversand constitutional symptoms persisted and on day 15, the patient hadtransient cardiac dysfunction: all symptoms resolved aftercorticosteroid therapy was initiated on day 18. Following CART19infusion, and coincident with the onset of high fevers, the patient hadrapid clearance of the p53-deficient CLL cells from peripheral blood asdepicted in FIG. 5A and a partial reduction of adenopathy, bone marrowshowed persistent extensive infiltration of CLL one month after therapydespite dramatic peripheral blood cytoreduction. The patient remainsasymptomatic.

Patient UPN 03 received pentostatin and cyclophosphamide aslymphodepleting chemotherapy prior to CART19 cell infusion. Three daysafter chemotherapy but prior to cell infusion, bone marrow washypercellular (60%) with approximately 50% involvement by CLL. Thepatient received a low dose of CART19 cells (1.5×10⁵ CAR+ T cells/kgdivided over 3 days). Again, there were no acute infusional toxicities.However, 14 days after the first infusion, the patient began havingrigors, fevers, nausea and diarrhea. By day 22 after infusion, tumorlysis syndrome was diagnosed requiring hospitalization. The patient hadresolution of constitutional symptoms, and within 1 month of CART19infusions, the patient had clearance of circulating CLL from the bloodand bone marrow by morphology, flow cytometry, cytogenetic, and FISHanalysis. CT scans showed resolution of abnormal adenopathy as depictedin FIGS. 5B and 5C. Complete remission was sustained beyond 8 monthsfrom the initial CART19 cell infusion.

Considerations of In Vivo CART19 Effector to CLL Target Cell Ratio

Pre-clinical studies showed that large tumors could be ablated, and thatthe infusion of 2.2×10⁷ CARs could eradicate tumors comprised of 1×10⁹cells, for an in vivo E:T ratio of 1:42 in humanized mice (Carpenito etal., 2009, Proc Natl Acad Sci USA 106:3360-3365), although thesecalculations did not take into account the expansion of T cells afterinjection. Estimation of CLL tumor burden over time permitted thecalculation of tumor reduction and the estimated CART19 E:T ratiosachieved in vivo in the three subjects based on number of CAR+ T cellsinfused. Tumor burdens were calculated by measuring CLL load in bonemarrow, blood and secondary lymphoid tissues. The baseline tumor burdensas shown in FIG. 10 indicate that each patient had on the order of 10¹²CLL cells (i.e. 1 kilogram tumor load) before CART19 infusion. PatientUPN 03 had an estimated baseline tumor burden of 8.8×10¹¹ CLL cells inthe bone marrow on day −1 (i.e. post chemotherapy and pre-CART19infusion), and a measured tumor mass in secondary lymphoid tissues of3.3-5.5×10¹¹ CLL cells, depending on the method of volumetric CT scananalysis. Given that UPN 03 was infused with only 1.4×10⁷ CART19 cells,using the estimate of initial total tumor burden (1.3×10¹² CLL cells),and that no CLL cells are detectable post treatment, a striking 1:93,000E:T ratio was achieved. By similar calculations, an effective E:T ratioin vivo of 1:2200 and 1:1000 was calculated for UPN 01 and UPN 02 asshown in Table 3). In the end, a contribution of serial killing byCART19 T cells, combined with in vivo CART19 expansion of >1,000-foldlikely contributed to the powerful anti-leukemic effects mediated byCART19 cells.

T Cells Expressing Chimeric Receptors Establish Memory and PotentAntitumor Effects in Patients with Advanced Leukemia

Limited in viva expression and effector function of CARs has been acentral limitation in the trials testing first generation CARs (Kershawet al., 2006, Clin Cancer Res 12:6106-6115; Lamers et al., 2006, J ClinOncol 24:e20-e22; Till et al., 2008, Blood, 112, 2261-2271; Park et al.,2007, Mol Ther 15:825833; Pule et al., 2008, Nat Med 14:1264-1270).Based on pre-clinical modeling demonstrating enhanced persistence ofCARs containing a 4-1BB signaling module (Milone et al., 2009, Mol Ther.17:1453-1464; Carpenito et al., 2009, Proc Natl Acad Sci USA106:3360-3365), experiments were designed to develop a second generationof CARs engineered with lentiviral vector technology. This secondgeneration of CARs was found to be safe in the setting of chronic HIVinfection (Levine et al., 2006, Proc Natl Acad Sci USA 103:17372-17377).The present results show that when this second generation CAR wasexpressed in T cells and cultured under conditions designed to promoteengraftment of central memory T cells (Rapoport et al., 2005, Nat Med11:1230-1237; Bondanza et al., 2006. Blood 107:1828-1836), improvedexpansion of CAR T cells after infusion was observed compared toprevious reports. CART19 cells established CD19-specific cellularmemory, and killed tumor cells at E:T ratios in vivo not previouslyachieved.

CART19 is the first CAR trial to incorporate a 4-1BB signaling domainand the first to use lentiviral vector technology. The present resultsdemonstrate efficient tracking of CARs to sites of tumor, with the defacto establishment of“tumor infiltrating lymphocytes” that exhibitedCD19 specificity. The pronounced in vivo expansion permitted the firstdemonstration that CARs directly recovered from patients can retaineffector function in vivo for months. A previous study had suggestedthat introduction of a first generation CAR into virus specific T cellsis preferable to primary T cells (Pule et al., 2008, Nat Med14:1264-1270), however the results with second generation CARsintroduced into optimally costimulated primary T cells calls this notioninto question. Without wishing to be bound by any particular theory, acautionary note is raised that the clinical effects were profound andunprecedented with the lysis of kilogram sized tumor burdens in allthree patients accompanied with the delayed release of potentiallydangerously high levels of cytokines in two of the patients. Classicalcytokine storm effects were not observed. However, the present study wasdesigned to mitigate this possibility by deliberate infusion of CART19over a period of three days.

It was found that very low doses of CARs can elicit potent clinicalresponses. This was a pilot study that demonstrated safety of the CART19vector design. The observation that doses of CART19 cells several ordersof magnitude below those tested in previous trials can have clinicalbenefit may have important implications for future implementation of CARtherapy on a wider scale, and for the design of trials testing CARsdirected against targets other than CD19.

The present studies further indicate that CART19 is expressed in bothcentral memory and effector T cells, and this likely contributes totheir long term survival compared to previous reports. Without wishingto be bound by any particular theory, CAR T cells may differentiate invivo into a central memory-like state upon encounter and subsequentelimination of target cells (e.g. CLL tumor cells or normal B cells)expressing the surrogate antigen. Indeed signaling of 4-1BB has beenreported to promote the development of memory in the context of TCRsignaling (Sabbagh et al., 2007, Trends Immunol 28:333-339).

The extended proliferation and survival of CART19 has revealed aspectsof the pharmacokinetics of CAR T cells that have not previously beenreported. It was observed that the kinetics of cytokine release in serumand marrow correlated with peak CART19 levels, so that it is possiblethat the decay is initiated when cellular targets expressing CD19 becomelimiting. The mechanism of the extended survival of CART19 may relate tothe aforementioned incorporation of the 4-1BB domain or to signalingthrough the natural TCR and/or CAR. An intriguing possibility is thatthe extended survival is related to the population of CART19 that hasbeen identified in marrow specimens, raising the hypothesis that CD19CARs could be maintained by encounter with B cell progenitors in thebone marrow. Related to this question is what drives the initialexpansion of CART19 cells in vivo? With rare exceptions (Savoldo et al.,2011, J Clin Invest doi: 10.1172/JCI46110; Pule et al., 2008, Nat Med14:1264-1270), the present study is the only trial to have omitted IL-2infusions, so that the CART19 cells likely either expanded in responseto homeostatic cytokines or more likely, to CD19 expressed on leukemictargets and/or normal B cells. In the latter case, this could be anattractive feature for CARs directed against targets on normal APCs suchas CD19 and CD20, as it is possible that self renewal of CART19 occurson the normal cells, providing a mechanism for CAR memory by means of“self vaccination/boosting” and therefore, long term tumorimmunosurveillance. The mechanisms of CART19 homeostasis may requirefurther study to elucidate cell intrinsic and extrinsic mechanisms ofpersistence. Previous to these results, most investigators have viewedCAR therapy as a transient form of immunotherapy, however CARs withoptimized signaling domains may have a role in both remission inductionand consolidation as well as for long term immunosurveillance.

Potent anti-leukemic effects have been observed in all three patients,including two patients with p53 deficient leukemia. Previous studieswith CARs have had difficulty separating antitumor effects fromlymphodepleting chemotherapy. However, the delayed cytokine releasecombined with the kinetics of tumor lysis in fludarabine-refractorypatients that was coincident, and possibly dependent on in vivo CARexpansion in the present study, indicate that CART19 mediates potentantitumor effects. The present results do not exclude a role forchemotherapy in potentiating the effects of CARs.

A thorough comparison of the vector, transgene and cell manufacturingprocedures with results from ongoing studies at other centers may berequired to gain a full understanding of the key features required toobtain sustained function of CAR T cells in vivo. Unlike antibodytherapies, CAR-modified T cells have the potential to replicate in vivo,and long-term persistence could lead to sustained tumor control. Theavailability of an off the shelf therapy comprised of non-crossresistant killer T cells has the potential to improve the outcome ofpatients with B cell malignancies. A limitation of antibody therapy, asfor example, with agents such as rituximab and bevicizumab, is that thetherapy requires repeated antibody infusions, that is inconvenient andcostly. The delivery of prolonged antibody therapy (in this case for atleast 6 months in 3 of 3 patients treated to date) with anti-CD19 scFvexpressed on T cells following a single infusion of CART19 cells has anumber of practical advantages, including conveniences and cost savings.

Example 2 Chimeric Antigen Receptor-Modified T Cells in Chronic LymphoidLeukemia

A lentiviral vector expressing a chimeric antigen receptor withspecificity for the B-cell antigen CD19, coupled with CD137 (acostimulatory receptor in T cells [4-1BB]) and CD3-zeta (asignal-transduction component of the T-cell antigen receptor) signalingdomains, was designed. It was observed that a low dose (approximately1.5×10⁵ cells per kilogram of body weight) of autologous chimericantigen receptor-modified T cells reinfused into a patient withrefractory chronic lymphocytic leukemia (CLL) expanded to a level thatwas more than 1000 times as high as the initial engraftment level invivo. It was also observed that the patient exhibited delayeddevelopment of the tumor lysis syndrome and with complete remission.

Apart from the tumor lysis syndrome, the only other grade ¾ toxic effectrelated to chimeric antigen receptor T cells was lymphopenia. Engineeredcells persisted at high levels for at least 6 months in the blood andbone marrow and continued to express the chimeric antigen receptor. Aspecific immune response was detected in the bone marrow, accompanied byloss of normal B cells and leukemia cells that express CD19. Remissionwas ongoing 10 months after treatment. Hypogammaglobulinemia was anexpected chronic toxic effect.

The materials and methods employed in these experiments are nowdescribed.

Materials and Methods

Study Procedures

A self-inactivating lentiviral vector (GeMCRIS 0607-793) was designed,which was subjected to preclinical safety testing, as reportedpreviously (Milone et al., 2009. Mol Ther, 17: 1453-64). Methods ofT-cell preparation have also been described previously (Porter et al,2006, Blood, 107:1325-31). Quantitative polymerase-chain-reaction (PCR)analysis was performed to detect chimeric antigen receptor T cells inblood and bone marrow. The lower limit of quantification was determinedfrom the standard curve; average values below the lower limit ofquantification (i.e., reportable but not quantifiable) are consideredapproximate. The lower limit of quantification of the assay was 25copies per microgram of genomic DNA.

Soluble-factor analysis was performed with the use of serum from wholeblood and bone marrow that was separated into aliquots for single useand stored at −80° C. Quantification of soluble cytokine factors wasperformed with the use of Luminex bead-array technology and reagents(Life Technologies).

Apheresis #1

A 12-15 liter apheresis procedure is carried out at the apheresiscenter. Peripheral blood mononuclear cells (PBMC) are obtained forCART-19 T cell generation during this procedure. From a singleleukapheresis, at least 50×10⁹ white blood cells are harvested tomanufacture CART-19 T cells. Baseline blood leukocytes are also obtainedand cryopreserved.

Cytroreductive Chemotherapy

Chemotherapy is started approximately 5-10 days before infusion so thatCART-19 cells may be given 1-2 days after completion of thechemotherapy. The timing of chemotherapy initiation therefore depends onthe length of the regimen. The purpose of the chemotherapy is to inducelymphopenia in order to facilitate engraftment and homeostatic expansionof CART-19 cells. The chemotherapy may be chosen also to reduce diseasetumor burden. The cytoreductive chemotherapy is chosen and administeredby community oncologists. The choice of chemotherapy depends on thepatients underlying disease and prior therapies. Fludarabine (30mg/m2/day×3 days) and cyclophosphamide (300 mg/m2/day×3 days) are theagents of choice, as there is the most experience with the use of theseagents in facilitating adoptive immunotherapy. Several other acceptableregimens using FDA-approved drugs are appropriate, including CHOP,HyperCVAD, EPOCH, DHAP, ICE or other regimens.

Restaging Assessment

A limited restaging is performed at the completion of chemotherapy inorder to provide baseline tumor burden measurements. This includesimaging, physical examination, and minimal residual disease (MRD)assessments. Subjects undergo the following for pre-infusing testing:physical exam, documentation of adverse events and blood draws forhematology, chemistry and pregnancy testing (if applicable).

Preparation of CART-19 T cells

Autologous T cells are engineered to express an extracellular singlechain antibody (scFv) with specificity for CD19. The extracellular scFvcan redirect specificity of the transduced T cells for cells thatexpress CD19, a molecule that is restricted in expression on the surfaceof the malignant cells and on normal B cells. In addition to CD19 scFv,the cells are transduced to express an intracellular signaling moleculecomprised of either the TCRζ chain or a tandem signaling domaincomprised of 4-1BB and TCRζ signaling modules. The scFv is derived froma mouse monoclonal antibody, and thus contains mouse sequences, and thesignaling domains are entirely of the native human sequences. CART-19 Tcells are manufactured by isolating the T cells by apheresis, and usinglentiviral vector technology (Dropulic et al., 2006, Human Gene Therapy,17: 577-88; Naldini et al., 1996, Science, 272: 263-7: Dull et al.,1998, J Virol, 72: 8463-71) to introduce the scFv:TCRζ:4-1BB into CD4and CD8 T cells. In some patients, a control scFv:TCRζ: is introducedinto a portion of the cells for a competitive repopulation experiment.These receptors are “universal” in that they bind antigen in anMHC-independent fashion, thus, one receptor construct can be used totreat a population of patients with CD19 antigen-positive tumors.

The CAR constructs were developed at the University of Pennsylvania, andthe clinical grade vector was manufactured at Lentigen Corporation. TheCART-19 cells are manufactured in the Clinical Cell and VaccineProduction Facility at the University of Pennsylvania according to theprocess shown in FIG. 11. At the end of cell cultures, the cells arecryopreserved in infusible cryomedia. A single dose of CART-19transduced T cells comprising of the infusion of 2.5×10⁹ to 5×10⁹ totalcells, are administered in either 1 or 2 bags. Each bag contains analiquot (volume dependent upon dose) of cryomedia containing thefollowing infusible grade reagents (% v/v): 31.25 plasmalyte-A, 31.25dextrose (5%), 0.45 NaCl, up to 7.50 DMSO, 1.00 dextran 40, 5.00 humanserum albumin with approximately 2.5-5×10⁹ autologous T cells per bag.For increased safety, the first dose is given as a split dose on days 0,1 and 2, with ˜10% of the cells on day 0, 30% on day 1, and 60% on day2.

Storage

Bags (10 to 100 ml capacity) containing CART-19-transduced T cells arestored in blood bank conditions in a monitored −135° C. freezer.Infusion bags are stored in the freezer until needed.

Cell Thawing

After logging the cells in the investigational pharmacy, frozen cellsare transported in dry ice to the subject's bedside. The cells arethawed at the bedside one bag at a time using a water bath maintained at36° C. to 38° C. The bag is gently massaged until the cells have justthawed. There should be no frozen clumps left in the container. If theCART-19 cell product appears to have a damaged or leaking bag, orotherwise appears to be compromised, it should not be infused.

Premedication

Side effects following T cell infusions may include transient fever,chills, and/or nausea. It is recommended that the subject bepre-medicated with acetaminophen 650 mg by mouth and diphenhydraminehydrochloride 25-50 mg by mouth or IV, prior to the infusion of CART-19cells. These medications may be repeated every six hours as needed. Acourse of non-steroidal anti-inflammatory medication may be prescribedif the patient continues to have fever not relieved by acetaminophen. Itis recommended that patients not receive systemic corticosteroids suchas hydrocortisone, prednisone, prednisolone (Solu-Medrol) ordexamethasone (Decadron) at any time, except in the case of alife-threatening emergency, since this may have an adverse effect on Tcells. If corticosteroids are required for an acute infusional reaction,an initial dose of hydrocortisone 100 mg is recommended.

Administration/Infusion

Infusions begin 1 to 2 days after completion of chemotherapy. The day ofthe first infusions, patients have a CBC with differential, andassessment of CD3, CD4 and CD8 counts since chemotherapy is given inpart to induce lymphopenia. Without wishing to be bound by anyparticular theory, it is believed that an initial i.v. dose of 2.5-5×10⁹CART-19 cells is optimal for this protocol. Because there are about1×10¹² T cells in a healthy adult, the proposed total dose is equivalentto about 0.5% of the total body mass of T cells (Roederer, 1995, NatMed, 1: 621-7; Macallan et al., 2003, Eur J Immunol, 33: 2316-26). Thefirst dose is administered using a split dose on days 0 (10%), 1 (30%)and 2 (60%). Subjects receive infusion in an isolated room. The cellsare thawed at the patient's bedside as described elsewhere herein. Thethawed cells are given at an infusion rate as quickly as tolerated sothat the duration of the infusion is approximately 10-15 minutes. Thetransduced T cells are administered by rapid intravenous infusion at aflow rate of approximately 10 mL to 20 mL per minute through an 18-gaugelatex free Y-type blood set with a 3-way stopcock. The duration of theinfusion is approximately 15 minutes. One or two bags of CART-19modified cells are delivered on ice, and the cells are administered tothe subject while cold. In subjects receiving mixtures of CART-19 cells,in order to facilitate mixing, the cells are administered simultaneouslyusing a Y-adapter. Subjects are infused and premedicated as describedelsewhere herein. Subjects' vital signs are assessed and pulse oximetryis done prior to dosing, at the end of the infusion and every 15 minutesthereafter for 1 hour and until these are stable and satisfactory. Ablood sample for determination of baseline CART-19 level is obtainedbefore infusion and 20 minutes post infusion. Patients experiencingtoxicities from their preceding cytoreductive chemotherapy have theirinfusion schedule delayed until these toxicities have resolved. Thespecific toxicities warranting delay of T cell infusions include: 1)Pulmonary: Requirement for supplemental oxygen to keep saturationgreater than 95% or presence of radiographic abnormalities on chestx-ray that are progressive; 2) Cardiac: New cardiac arrhythmia notcontrolled with medical management. 3) Hypotension requiring pressorsupport. 4) Active Infection: Positive blood cultures for bacteria,fungus, or virus within 48-hours of T cell infusion. A serum sample forpotassium and uric acid is collected before the first infusion as wellas two hours after each subsequent infusion.

Post Infusion Laboratories to Assess Graftment and Persistence

Subjects return at day 4 and 10 after the initial CART-19 cell infusionto have blood drawn for serum cytokine levels, and CART-19 PCR in orderto evaluate the presence of CART-19 cells. Subjects return once a weekfor three weeks to undergo the following: physical exam, documentationof adverse events and blood draws for hematology, chemistry, engraftmentand persistence of CART-19 cells and research labs.

Second Infusion

Without wishing to be bound by any particular theory, it is believedthat a second dose of CART-19 cells can be given on day 11 to patients,provided that they exhibit adequate tolerance to the first dose andsufficient CART-19 cells were manufactured. The dose is 2-5×10⁹ totalcells. A serum sample for potassium and uric acid can be collected twohours after the infusion.

Second Apheresis

A 2 liter apheresis procedure is carried out at the apheresis center.PBMC are obtained for research and cryopreserved. Subjects undergo thefollowing: physical exam, documentation of adverse events and blooddraws for hematology, chemistry, engraftment and persistence of CART-19cells and research labs. In addition restaging is done in order toprovide tumor burden measurements. Restaging testing is determined bydisease type and includes imaging, MRD assessments, bone marrow aspirateand biopsy and/or lymph node biopsy.

Monthly Evaluations 2 to 6 Months Post Infusion

Subjects return on a monthly basis during months 2 to 6 post CART-19cell infusion. At these study visits, subjects undergo the following:concomitant medication, physical exam, documentation of adverse eventsand blood draws for hematology, chemistry, engraftment and persistenceof CART-19 cells and research labs. The HIV DNA assay is performed atmonths 2-6 post CART-19 cell infusion to exclude the presence ofdetectable RCL.

Quarterly Evaluations Up to 2 Years Post Infusion

Subjects are evaluated on a quarterly basis until 2 years post infusion.At these study visits, subjects undergo the following: concomitantmedication, physical exam, documentation of adverse events and blooddraws for hematology, chemistry, engraftment and persistence of CART-19cells and research labs. The HIV DNA assay is performed at months 3 and6 post CART-19 cell infusion to exclude the presence of detectable RCL.

The results of the experiments are now described

Patient History

The patient received a diagnosis of stage I CLL in 1996. He firstrequired treatment after 6 years of observation for progressiveleukocytosis and adenopathy. In 2002, he was treated with two cycles ofrituximab plus fludarabine; this treatment resulted in normalization ofblood counts and partial resolution of adenopathy. In 2006, he receivedfour cycles of rituximab and fludarabine for disease progression, againwith normalization of blood counts and partial regression of adenopathy.This response was followed by a 20-month progression-free interval and a2-year treatment-free interval. In February 2009, he had rapidlyprogressive leukocytosis and recurrent adenopathy. His bone marrow wasextensively infiltrated with CLL. Cytogenetic analysis showed that 3 of15 cells contained a deletion of chromosome 17p, and fluorescence insitu hybridization (FISH) testing showed that 170 of 200 cells had adeletion involving TP53 on chromosome 17p. He received rituximab withbendamustine for one cycle and three additional cycles of bendamustinewithout rituximab (because of a severe allergic reaction). Thistreatment resulted in only transient improvement in lymphocytosis.Progressive adenopathy was documented by means of computed tomography(CT) after therapy.

Autologous T cells were collected by means of leukapheresis andcryopreserved. The patient then received alemtuzumab (an anti-CD52,mature-lymphocyte, cell-surface antigen) for 11 weeks, with improvedhematopoiesis and a partial resolution of adenopathy. Over the next 6months, he had stable disease with persistent, extensive marrowinvolvement and diffuse adenopathy with multiple 1- to 3-cm lymph nodes.In July 2010, the patient was enrolled in a phase 1 clinical trial ofchimeric antigen receptor-modified T cells.

Cell Infusions

Autologous T cells from the patient were thawed and transduced withlentivirus to express the CD19-specific chimeric antigen receptor (FIG.12A); sequence identifiers for the lentiviral vector and relevantsequences are depicted in Table 5. Four days before cell infusion, thepatient received chemotherapy designed for depletion of lymphocytes(pentostatin at a dose of 4 mg per square meter of body-surface area andcyclophosphamide at a dose of 600 mg per square meter) without rituximab(Lamanna et al., 2006, J Clin Oncol, 24: 1575-81). Three days afterchemotherapy but before cell infusion, the bone marrow was hypercellularwith approximately 40% involvement by CLL. Leukemia cells expressedkappa light chain and CD5, CD19, CD20, and CD23. Cytogenetic analysisshowed two separate clones, both resulting in loss of chromosome 17p andthe TP53 locus (46, XY, del(17)(p 12)[5]/46, XY,der(17)t(1721)(q10:q10)[5]/46, XY[14]). Four days after chemotherapy,the patient received a total of 3×10⁸ T cells, of which 5% weretransduced, for a total of 1.42×10⁷ transduced cells (1.46×10⁵ cells perkilogram) split into three consecutive daily intravenous infusions (10%on day 1, 30% on day 2, and 60% on day 3). No postinfusion cytokineswere administered. No toxic effects of infusions were noted.

TABLE 5 Sequence identifiers for pELPS-CD19-BBz transfer vector SEQ IDNO: # IDENTITY SEQ ID NO: 1 pELPS-CD19-BBZ transfer vector (nucleic acidsequence) SEQ ID NO: 2 RSV's U3 (nucleic acid sequence) SEQ ID NO: 3 HIVR repeat (nucleic acid sequence) SEQ ID NO: 4 HIV U5 Repeat (nucleicacid sequence) SEQ ID NO: 5 Partial Gag/Pol (nucleic acid sequence) SEQID NO: 6 cPPT (nucleic acid sequence) SEQ ID NO: 7 EF1 alpha Promoter(nucleic acid sequence) SEQ ID NO: 8 CD19-BBzeta CAR (nucleic acidsequence) SEQ ID NO: 9 Hu Woodchuck PRE (nucleic acid sequence) SEQ IDNO: 10 R Repeat (nucleic acid sequence)t SEQ ID NO: 11 U5 Repeat(nucleic acid sequence) SEQ ID NO: 12 CD19-BBzeta CAR (amino acidsequence) SEQ ID NO: 13 CD8 Leader (nucleic acid sequence) SEQ ID NO: 14Anti-CD19scFv (nucleic acid sequence) SEQ ID NO: 15 CD8 Hinge (nucleicacid sequence) SEQ ID NO: 16 CD8 Transmembrane (nucleic acid sequence)SEQ ID NO: 17 4-1BB (nucleic acid sequence) SEQ ID NO: 18 CD3zeta(nucleic acid sequence) SEQ ID NO: 19 CD8 Leader (amino acid sequence)SEQ ID NO: 20 Anti-CD19scFv (amino acid sequence) SEQ ID NO: 21 CD8Hinge (amino acid sequence) SEQ ID NO: 22 CD8 Transmembrane (amino acidsequence) SEQ ID NO: 23 4-1BB (amino acid sequence) SEQ ID NO: 24CD3zeta (amino acid sequence)

Clinical Response and Evaluations

Fourteen days after the first infusion, the patient began having chillsand low-grade fevers associated with grade 2 fatigue. Over the next 5days, the chills intensified, and his temperature escalated to 39.2° C.(102.5° F.), associated with rigors, diaphoresis, anorexia, nausea, anddiarrhea. He had no respiratory or cardiac symptoms. Because of thefevers, chest radiography and blood, urine, and stool cultures wereperformed, and were all negative or normal. The tumor lysis syndrome wasdiagnosed on day 22 after infusion (FIG. 12B). The uric acid level was10.6 mg per deciliter (630.5 μmol per liter), the phosphorus level was4.7 mg per deciliter (1.5 mmol per liter) (normal range, 2.4 to 4.7 mgper deciliter [0.8 to 1.5 mmol per liter]), and the lactatedehydrogenase level was 1130 U per liter (normal range, 98 to 192).There was evidence of acute kidney injury, with a creatinine level of2.60 mg per deciliter (229.8 μmol per liter) (baseline level, <1.0 mgper deciliter [<88.4 μmol per liter]). The patient was hospitalized andtreated with fluid resuscitation and rasburicase. The uric acid levelreturned to the normal range within 24 hours, and the creatinine levelwithin 3 days; he was discharged on hospital day 4. The lactatedehydrogenase level decreased gradually, becoming normal over thefollowing month.

By day 28 after CART19-cell infusion, adenopathy was no longer palpable,and on day 23, there was no evidence of CLL in the bone marrow (FIG.12C). The karyotype was now normal in 15 of 15 cells (46, XY), and FISHtesting was negative for deletion TP53 in 198 of 200 cells examined;this is considered to be within normal limits in negative controls.Flow-cytometric analysis showed no residual CLL, and B cells were notdetectable (<1% of cells within the CD5+CD10-CD19+CD23+ lymphocytegate). CT scanning performed on day 31 after infusion showed resolutionof adenopathy (FIG. 12D).

Three and 6 months after CART19-cell infusion, the physical examinationremained unremarkable, with no palpable adenopathy, and CT scanningperformed 3 months after CART19-cell infusion showed sustained remission(FIG. 12D). Bone marrow studies at 3 and 6 months also showed noevidence of CLL by means of morphologic analysis, karyotype analysis(46, XY), or flow-cytometric analysis, with a continued lack of normal Bcells as well. Remission had been sustained for at least 10 months.

Toxicity of CART19 Cells

The cell infusions had no acute toxic effects. The only serious (grade 3or 4) adverse event noted was the grade 3 tumor lysis syndrome describedabove. The patient had grade 1 lymphopenia at baseline and grade 2 or 3lymphopenia beginning on day 1 and continuing through at least 10 monthsafter therapy. Grade 4 lymphopenia, with an absolute lymphocyte count of140 cells per cubic millimeter, was recorded on day 19, but from day 22through at least 10 months, the absolute lymphocyte count ranged between390 and 780 cells per cubic millimeter (grade 2 or 3 lymphopenia). Thepatient had transient grade 1 thrombocytopenia (platelet count, 98,000to 131,000 per cubic millimeter) from day 19 through day 26 and grade 1or 2 neutropenia (absolute neutrophil count, 1090 to 1630 per cubicmillimeter) from day 17 through day 33. Other signs and symptoms thatwere probably related to the study treatment included grade 1 and 2elevations in aminotransferase and alkaline phosphatase levels, whichdeveloped 17 days after the first infusion and resolved by day 33. Grade1 and 2 constitutional symptoms consisted of fevers, chills,diaphoresis, myalgias, headache, and fatigue. Grade 2hypogammaglobulinemia was corrected with infusions of intravenous immuneglobulin.

Analysis of Serum and Bone Marrow Cytokines

The patient's clinical response was accompanied by a delayed increase inlevels of inflammatory cytokines (FIG. 13A through FIG. 13D), withlevels of interferon-γ, the interferon-γ-responsive chemokines CXCL9 andCXCL10, and interleukin-6 that were 160 times as high as baselinelevels. The temporal rise in cytokine levels paralleled the clinicalsymptoms, peaking 17 to 23 days after the first CART19-cell infusion.

The supernatants from serial bone marrow aspirates were measured forcytokines and showed evidence of immune activation (FIG. 13E).Significant increases in interferon-γ, CXCL9, interleukin-6, and solubleinterleukin-2 receptor were noted, as compared with the baseline levelson the day before T-cell infusion; the values peaked on day 23 after thefirst CART19-cell infusion. The increase in bone marrow cytokinescoincided with the elimination of leukemia cells from the marrow. Serumand marrow tumor necrosis factor α remained unchanged.

Expansion and Persistence of Chimeric Antigen Receptor T Cells

Real-time PCR detected DNA encoding anti-CD19 chimeric antigen receptor(CAR19) beginning on day 1 after the first infusion (FIG. 14A). Morethan a 3-log expansion of the cells in vivo was noted by day 21 afterinfusion. At peak levels, CART19 cells in blood accounted for more than20% of circulating lymphocytes; these peak levels coincided with theoccurrence of constitutional symptoms, the tumor lysis syndrome (FIG.12B), and elevations in serum cytokine levels (FIG. 13A through FIG.13D). CART19 cells remained detectable at high levels 6 months after theinfusions, though the values decreased by a factor of 10 from peaklevels. The doubling time of chimeric antigen receptor T cells in bloodwas approximately 1.2 days, with an elimination half-life of 31 days.Chimeric Antigen Receptor T Cells in Bone Marrow CART19 cells wereidentified in bone marrow specimens beginning 23 days after the firstinfusion (FIG. 14B) and persisted for at least 6 months, with a decayhalf-life of 34 days. The highest levels of CART19 cells in the bonemarrow were identified at the first assessment 23 days after the firstinfusion and coincided with induction of an immune response, asindicated by cytokine-secretion profiles (FIG. 13E). Flow-cytometricanalysis of bone marrow aspirates indicated a clonal expansion of CD5+CD19+ cells at baseline that was absent 1 month after infusion and in asample obtained 3 months after infusion (data not shown). Normal B cellswere not detected after treatment (FIG. 14C).

Treatment with Autologous Genetically Modified CART19 Cells

Described herein is the delayed development of the tumor lysis syndromeand a complete response 3 weeks after treatment with autologous T cellsgenetically modified to target CD19 through transduction with alentivirus vector expressing anti-CD19 linked to CD3-zeta and CD137(4-1BB) signaling domains. Genetically modified cells were present athigh levels in bone marrow for at least 6 months after infusion. Thegeneration of a CD19-specific immune response in bone marrow wasdemonstrated by temporal release of cytokines and ablation of leukemiacells that coincided with peak infiltration of chimeric antigen receptorT cells. Development of the tumor lysis syndrome after cellularimmunotherapy has not been reported previously (Baeksgaard et al., 2003,Cancer Chemother Pharacol, 51: 187-92).

Genetic manipulation of autologous T cells to target specific tumorantigens is an attractive strategy for cancer therapy (Sadelain et al.,2009, Curr Opin Immunol, 21: 215-23; Jena et al., 2010, Blood, 116:1035-44). An important feature of the approach described herein is thatchimeric antigen receptor T cells can recognize tumor targets in anHLA-unrestricted manner, so that “off-the-shelf” chimeric antigenreceptors can be constructed for tumors with a wide variety ofhistologic features. HIV-derived lentiviral vectors were used for cancertherapy, an approach that may have some advantages over the use ofretroviral vectors (June et al., 2009, Nat Rev Immunol, 9: 704-16).

In previous trials of chimeric antigen receptor T cells, objective tumorresponses have been modest, and in vivo proliferation of modified cellshas not been sustained (Kershaw et al., 2006, Clin Cancer Res, 12:6106-15; Till et al., 2008, Blood, 112: 2261-71; Pule et al., 2008, NatMed, 14: 1264-70). Brentjens and colleagues reported preliminary resultsof a clinical trial of CD19-targeted chimeric antigen receptors linkedto a CD28 signaling domain and found transient tumor responses in two ofthree patients with advanced CLL (Brentjens et al., 2010, Mol Ther, 18:666-8); however, the chimeric antigen receptors rapidly disappeared fromthe circulation.

It was unexpected that the very low dose of chimeric antigen receptor Tcells that were infused would result in a clinically evident antitumorresponse. Indeed, the infused dose of 1.5×10⁵ chimeric antigen receptorT cells per kilogram was several orders of magnitude below doses used inprevious studies of T cells modified to express chimeric antigenreceptors or transgenic T-cell receptors (Kershaw et al., 2006, ClinCancer Res, 12: 6106-15; Brentjens et al., 2010, Mol Ther, 18: 666-8;Morgan et al., 2010. Mol Ther, 18: 843-51; Johnson et al., 2009, Blood,114: 535-46). Without being held to any particular theory, it isspeculated that the chemotherapy may potentiate the effects of chimericantigen receptor.

The prolonged persistence of CART19 cells in the blood and bone marrowof the patient results from inclusion of the 4-1BB signaling domain. Itis likely that the CART19-cell-mediated elimination of normal B cellsfacilitated the induction of immunologic tolerance to the chimericantigen receptor, since the CART19 cells that express the single-chainFv antibody fragment containing murine sequences were not rejected.Given the absence of detectable CD19-positive leukemia cells in thispatient, and without being held to any particular theory, it is possiblethat homeostasis of the chimeric antigen receptor T cells was achievedat least in part from stimulation delivered by early B-cell progenitorsas they began to emerge in the bone marrow. The invention relates to thediscovery that a new mechanism may exist to maintain “memory” chimericantigen receptor T cells.

Although CD19 is an attractive tumor target, with expression limited tonormal and malignant B cells, there is concern that persistence of thechimeric antigen receptor T cells may mediate long-term B-celldeficiency. In fact, in the patient, B cells were absent from the bloodand bone marrow for at least 6 months after infusion. This patient didnot have recurrent infections. Targeting B cells through CD20 withrituximab is an effective and relatively safe strategy for patients withB-cell neoplasms, and long-term B-cell lymphopenia is manageable(Molina, 2008, Ann Rev Med, 59: 237-50). Patients treated with rituximabhave been reported to have a return of B cells within months afterdiscontinuation of therapy. It is not yet clear whether such recoveryoccurs in patients whose anti-B-cell T cells persist in vivo.

Patients who have CLL with TP53 deletions have short remissions afterstandard therapies (Dohner et al., 1995, Blood, 85: 1580-9). Allogeneicbone marrow transplantation has been the only approach that has inducedlong-term remissions in patients with advanced CLL (Gribben et al.,2011, Biol Blood Marrow Transplant, 17: Suppl:S63-S70). However, theresulting potent graft-versus-tumor effect is associated withconsiderable morbidity because of the high frequency of chronicgraft-versus-host disease, which is often especially severe in olderpatients—those who are typically affected by CLL (Gribben et al., 2011,Biol Blood Marrow Transplant, 17: Suppl:S63-S70; Sorror et al., 2008,Blood, 111: 446-52). The data presented herein suggests that geneticallymodified autologous T cells may circumvent this limitation.

The delayed onset of the tumor lysis syndrome and cytokine secretion,combined with vigorous in vivo chimeric antigen receptor T-cellexpansion and prominent antileukemia activity, points to substantial andsustained effector functions of the CART19 cells. Experiments describedherein highlights the potency of this therapy and provides support forthe detailed study of autologous T cells genetically modified to targetCD19 (and other targets) through transduction of a chimeric antigenreceptor linked to potent signaling domains. Unlike antibody-mediatedtherapy, chimeric antigen receptor-modified T cells have the potentialto replicate in vivo, and long-term persistence could lead to sustainedtumor control. Two other patients with advanced CLL have also receivedCART19 infusions according to this protocol, and all three have hadtumor responses. These findings warrant continued study ofCD19-redirected T cells for B-cell neoplasms.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

1-89. (canceled)
 90. A human T cell comprising a nucleic acid sequenceencoding a chimeric antigen receptor (CAR), wherein the CAR comprises aCD19 antigen binding domain comprising, from the amino to the carboxyterminus, a light chain variable region and a heavy chain variableregion of SEQ ID NO:20, wherein the CAR further comprises atransmembrane domain, a 4-1BB costimulatory signaling region, and a CD3zeta signaling domain.
 91. The human T cell of claim 90, wherein the Tcell from a human having a cancer.
 92. The human T cell of claim 90,wherein said antigen binding fragment is a scFv.
 93. The human T cell ofclaim 92, wherein the scFv comprises the amino acid sequence of SEQ IDNO:20.
 94. The human T cell of claim 90, wherein the transmembranedomain is CD8α transmembrane domain.
 95. The human T cell of claim 94,wherein the CD8α transmembrane domain comprises the amino acid sequenceof SEQ ID NO:
 22. 96. The human T cell of claim 90, wherein the CARfurther comprises a hinge domain.
 97. The human T cell of claim 96,wherein the hinge domain is a CD8α hinge domain.
 98. The human T cell ofclaim 97, wherein the CD8α hinge domain comprises the amino acidsequence of SEQ ID NO:21.
 99. The human T cell of claim 90, wherein the4-1BB costimulatory signaling region comprises the amino acid sequenceof SEQ ID NO:23.
 100. The human T cell of claim 90, wherein the CD3 zetasignaling domain comprises the amino acid sequence of SEQ ID NO: 24.101. The human T cell of claim 90, wherein the CD19 antigen bindingdomain is encoded by a nucleic acid sequence comprising SEQ ID NO: 14.102. The human T cell of claim 95, wherein the CD8α transmembrane domainis encoded by a nucleic acid sequence comprising SEQ ID NO:
 16. 103. Thehuman T cell of claim 98, wherein the CD8α hinge domain is encoded by anucleic acid sequence comprising SEQ ID NO:
 15. 104. The human T cell ofclaim 99, wherein the 4-1BB costimulatory signaling region is encoded bya nucleic acid sequence comprising SEQ ID NO:
 17. 105. The human T cellof claim 100, wherein the CD3 zeta signaling domain is encoded by anucleic acid sequence comprising SEQ ID NO:
 18. 106. The human T cell ofclaim 90, wherein the CAR comprises the amino acid sequence of SEQ IDNO:12.
 107. The human T cell of claim 106, wherein the CAR is encoded bya nucleic acid sequence comprising SEQ ID NO:8.
 108. The human T cell ofclaim 90, wherein the CAR further comprises a CD28 costimulatorysignaling region.
 109. The human T cell of claim 91, wherein the canceris a hematological cancer.
 110. The human T cell of claim 90, whereinthe T cell comprises a vector that comprises the nucleic acid sequence.111. The human T cell of claim 110, wherein the vector is a lentiviralvector.
 112. The human T cell of claim 111, wherein the vector furthercomprises a promoter.
 113. The human T cell of claim 112, wherein thepromoter is an EF-1α promoter.
 114. The human T cell of claim 90,wherein the cell is isolated from a blood sample obtained from a humanbefore the human is treated with a modality selected from the groupconsisting of an antiviral agent, chemotherapy, radiation, animmunosuppressive agent and an antibody.
 115. The human cell of claim114, wherein the antibody is selected from the group consisting ofanti-CD3 antibody, natalizumab and efalizumab.
 116. The human cell ofclaim 114, wherein the immunosuppressive agent is selected from thegroup consisting of cyclosporin, azathioprine, methotrexate,mycophenolate, FK506, CAMPATH, cytoxan, fludarabine, cyclosporin, FK506,rapamycin, mycophenolic acid, a steroid, and FR901228.